Standard Crive Lab Report

Standard Crive Lab Report
Purpose:
1. Assay a series of dilutions of known concentration and prepare the standard curve.
2. Determine the concentration of unknown 1 and unknown 2 based on the standard curve.
Methods:
I. Preparation of Diluted Bovine Serum Albumin (BSA) Standards
Five 1.5–ml tubes were prepared and labeled as 1 – 5. Guided by Table 1, agents were added in
order. Respectively pipetted 325 μl of BSA Stock and 325 μl PBS into tube 1. Mixed the solution by
pipetting. Pipetted 325 μl of tube 1 solution into tube 2, and added 325 μl of PBS into tube 2. Mixed
the solution by pipetting. Pipetted 325 μl of tube 2 solution into tube 3, and added 325 μl of PBS
into tube 3. Mixed the solution by pipetting. Pipetted 325 μl of tube 3 solution into tube 4, ... Show
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The coefficient of variation was expressed as a percentage.
Conclusions:
The standard curve is used to determine the concentration of each unknown sample. The trendline in
Figure 1 has a desired regression value: R2 = 1. R2 measures the distance between the fitted line and
the data points. The value ranges from 0 to 1, and the higher it is, the better the model fits the data
points. The value 1 suggests that the model is the best–fit model in which all the variability of data
around their mean can be explained. Therefore, the regression value is acceptable and the standard
curve is reliable.
For each unknown sample, the SD value and CV percentage is low.
Applications:
The BCA method can be applied to many research areas. During the assessment on the caries–
associated virulence (production of acid, extracellular polysaccharides, proteins and metabolic
activity) biofilms from Candida
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How To Write An Enzyme Lab Report
Enzymes are a diverse category of proteins that are used in various biochemical systems to catalyze
a particular reaction. They are vital in increasing the rate of reactions by up to 1017 which aids in
the functioning of cells while having implications in medicine, biotechnology, and the
food/pharmaceutical industries.1 The purpose of this experiment was to extract and purify an
enzyme of interest known as acid phosphatase. The process of enzyme isolation is essential for
using these proteins outside of a cell, unlocking their benefits for practical uses. However, the
procedure involves various steps with inherent risk of denaturing the enzyme via common
biochemical such as inhibitors and oxidizing/proteolytic agents, thus the effectiveness of the enzyme
is analyzed by taking samples (aliquots) at each purification step (fraction).1 The enzyme of interest
in this experiment was initially extracted from wheat germ via osmotic rupturing of the cellular
membrane to release the water–soluble protein.1 This solution was then centrifuged to sediment
various cellular debris into a pellet, leaving a solution of various hydrophilic solutes, including acid
phosphates. ... Show more content on Helpwriting.net ...
This procedure involves exposing the enzyme to two concentrations of the salt (35% and 57%) in
two different steps. This is called salt fractionation and effectively separates large groups of
proteins, lipids, carbohydrates, and DNA. The process works by causing the highly hydrophobic
molecules to become insoluble at 35% and the partially hydrophobic (acid phosphatase) to
precipitate at 57%, leaving the most hydrophilic solutes in solution.1 The salt concentration and type
may be varied based on the isolation compound of interest, for the overlying principle of this
technique, selective dehydration, remains the

Advantages And Disadvantages Of Dihydropyrimidin-2
Recently, the synthetic chemists have gradually taken concern of the compounds containing nucleus
of 3,4–dihydropyrimidin–2(1H)–one, which have a series of pharmacological characteristics, such
as calcium channels blockers, antioxidant, anticancer, and antiinﬂammatory activities [1–5]. But
their efficiently utilizing is boxed in low water solubility. The sulfonate group is strongly acidic. As
a result, it is expected that the solubility of 3,4–dihydropyrimidin–2(1H)–one derivatives could be
greatly improved by introducing the sulfonte group. In this essay, through sulphonation with good
hydrosoluble prosperities, we have successfully obtained two creative 3,4–dihydropyrimidin–
2(1H)–one derivatives 1, 2 (structures shown in Scheme 1).
Serum albumin (SA) is one of the most ... Show more content on Helpwriting.net ...
The reaction scheme is showed in Scheme 1. Brieﬂy, using sulfamic acid as a catalyst, DHPMs1 and
DHPMs2 were synthesized by ethanol thermal method. Urea (0.90 g, 15 mmol), acetaldehyde (0.44
g, 10 mmol) / benzaldehyde (1.06 g, 10 mmol), ethyl acetoacetate (1.56 g, 12 mmol) and sulfamic
acid (0.8 g, 8 mmol) were dissolved in 10 mL ethanol to form a clear solution, which was settled in
a 25 mL teflon–sealed autoclave and maintaining at 80 ºC for 2 h. After cooling, the reaction
mixture was cleaned with cold water and 50 % ethanol, and the residue recrystallized from 95 %
ethanol to afford the pure product. DHPMs1: Slightly water–soluble white powder; Yield = 1.31 g
(71 %); Mp 192 – 193; formula: C9H14N2O3; FW: 198.22; IR (KBr) cm–1：3244, 3118, 2978,
1702, 1654；ESI–MS (m/z): 197.33 (M + H). DHPMs2: Slightly water–soluble white powder;
Yield = 2.43 g (93.6 %); Mp 206 – 207; formula: C14H16N2O3; FW: 260.29; IR (KBr) cm–1：
3235, 3108, 2975, 1702, 1645；ESI–MS (m/z): 259.18 (M +

Paper Chromatography Lab Report
Paper Chromatography and Spectrophotometry Used to Identify Amino Acids and Concentration of
Protein in Solution
Introduction: Paper chromatography was used to characterize known amino acids in solution with an
accordant Rf value. These Rf values were then used to identify a solution with unknown amino acids
and observed Rf values. An Rf value is calculated using the following formula:Distance Traveled by
solute (cm)Distance Traveled by solvent (cm), where the distance traveled by solute is measured
from the origin line to the center of the solute spot (Lombard, p19). A polar matrix, composed of
cellulose, was utilized in tandem with a nonpolar solvent (Ammonia:Ethanol:dH2O) during paper
chromatography (Freeman, et al. p113). This diversity ... Show more content on Helpwriting.net ...
Unknown Y had two observed Rf values of 0.305 and 0.634. The primary Rf value deviated only
0.001 from the calculated Rf of Histidine while the secondary amino acid deviated 0.036 from the
nearest Rf value of 0.598 for Valine. The percent error for this set of Rf values is only 6.02%, which
we find to be within reasonable ranges. Despite the more significant error present in the secondary
amino acid identification, Unknown Y is still believed to be a solution of Histidine and Valine.
Unknown X had two observed Rf values of 0.147 and 0.529; of which, only the latter devotes form
a calculated amino acid Rf value. The former Rf matches exactly to Aspartic Acid while the latter
deviates 0.005 from the accepted Proline value. Given this close adherence to calculated values,
Unknown X is hypothesized to be a solution of Aspartic Acid and Proline. This is concurrent with
the stated hypothesis as charged amino acids such as aspartic Acid, Histidine, and Lysine had Rf
values ranging from 0.147 to 0.306 while those acids with nonpolar tendencies reported values from
0.524 to 0.776 (Freeman, et al. p80). The average value of polar/charged amino acids was 0.228,
which was 0.405 less than the average value of non polar groups 0.633. Clearly, the nonpolar amino
acids were interacting with the matrix in a way that decreased their travel distance over 1 hour,
therein supporting our hypothesis that adsorptive behavior will result in an increase Rf value relative
to amino acids with polar or charged R–groups. The second procedure performed was two parts
wherein we both optimized the wavelength for spectrophotometry and established a standard curve.
We established an absorption spectrum based on the incremental increase in wavelength by 10 nm
from 490 nm to 570 nm

Sam Case Study
Viperitoxin decreased the twitch response by around 52–53% and decreased the KCl response by
around 113%. There was no CCh response as it was not tested. Taipoxin decreased the twitch
response by 83%, whilst the CCh response did not change, but increased the twitch response of KCl
by 18%. For α–bungarotoxin, the twitch response decreased by 97%, whereas the CCh response by
decreased 84% and the KCl response decreased by 19%. Discussion of results 1. What is the
relevance of the "BSA" data and why is it important to include these data when making conclusions
about the mechanisms of action of the 3 toxins? A: The Bovine Albumin Serum 'BSA' is a protein
derived from cows and is commonly utilised in ELISA tests to prevent non–specific binding from
occurring. Conversely, it also serves as a protein supplement. The BSA is ... Show more content on
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Based on your conclusions, which, if any, of the venoms would likely cause a paralysis similar to
what Sam experienced when he was bitten by the snake. Explain your reasoning. This study does
not conclude which venom is responsible for Sam's paralysis, when bitten; Sam experienced
symptoms such as flaccid paralysis. Any of the three venoms tested (Viperitoxin, A–bungarotoxin
and Taipoxin) could cause similar levels of paralysis since all three toxins affect muscle contraction;
such as the post–synapse, pre–synapse or directly on the muscle. It is difficult to pin–point the
specific neurotoxin due to the similarity of causation of the symptom of flaccid paralysis. 4. What
information does this study provide about the reversibility of each of the toxins? Explain your
response. This study does not provide adequate information on the reversibility of the three toxins
presented. There is no data for each of the toxins on the twitch responses–post washing the toxin out
of the organ bath. Omitting this data inhibits the ability for this study to reach a conclusion, in terms
of finding out on whether there are reversibility qualities of each

Sds Page (Sodium Dodecyl Sulfate Polyacrylamide Gel...
Gel electrophoresis is a routine laboratory procedure in biochemical studies that takes advantage of
a protein's amphoteric nature to determine its molecular weight and charge by running the sample
through a gel matrix under the influence of an electrically charged field. A popular example of gel
electrophoresis is Sodium dodecyl sulfate–polyacrylamide electrophoresis or SDS– PAGE which
has been used in this experiment to supposedly determine albumin and casein's molecular weights
respectively. The system used in gel electrophoresis consists of 3 major parts: a stationary phase, a
mobile phase) and an electrical power supply.
In SDS–PAGE the polyacrylamide gel serves as the stationary phase, the buffer (Tris–HCl in this
case) as the ... Show more content on Helpwriting.net ...
On the other hand, the Tris–HCl buffer and deionized water provides the system its shape since the
polyacrylamide complex is just a thin film of interlocking polymer fibers without any liquid to trap
within.
Polyacrylamide gels are usually preferred over other gel types such as agarose in protein
electrophoresis because of various benefits such as its ability to be cast in various pore sizes which
provides options for the researchers who observe protein samples with various Kda's depending on
how separated they aim the bands to be or how well the sample is sieved. It is also preferred because
of its charge neutrality upon setting as it does not interfere with the electrophoresis which is highly
reliant on the sample and the surrounding's charges within the system. Lastly, the gel itself is
colorless and protein stains do not adhere to the gel so there will be minimal problems with
unnecessary stains that aren't from the staining solution.
APS and TEMED was used to initiate the binding of acrylamide to bisacrylamide via a vinyl
addition reaction where TEMED catalyzes the decomposition of APS into free sulfide molecules
which then initiates the formation of the polyacrylamide complex.
Sodium dodecyl–sulfate or SDS is a protein denaturant that destroys weak interactions within the
protein molecule which unfolds it

Synovial Bacterial Lab
Edwanny Nivar
Professor Fair
Microbial Physiology and Genetics
12 November 2017
Role of phenol–soluble modulins in formation of Staphylococcus aureus biofilms in synovial fluid
ABSTRACT/OVERVIEW QUESTIONS:
1. Who did the research, and where?
a. This research was done by Sana S. Dastgheyb, Amer E. Villaruz, Katherine Y. Le, Vee Y. Tan,
Anthony C. Duong, Som S. Chatterjee, Gordon Y. C. Cheung, Hwang–Soo Joo, Noree J. Hickok and
Michael Otto.
b. It was conducted at the Pathogen Molecular Genetics Section, Laboratory of Human Bacterial
Pathogenesis, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland, USA; and at the Department of Orthopedic Surgery, Thomas Jefferson
University, ... Show more content on Helpwriting.net ...
a. Staphylococcus aureus is a major cause of septic arthritis and orthopedic infection, in particular
those developing on prosthetic joints after arthroplasty. Joint infections can cause prolonged
disability and increased health care cost, due to prolonged antibiotic treatments, multiple surgeries,
and in some cases joint fusion. If there is rejection of treatment, these infections can cause
significant morbidity, including loss of limbs, systemic infection, and even death.
b. Biofilms are surface–attached bacterial agglomerations that frequently develop on indwelling
medical devices.
c. The matrix that connects cells in a biofilm consists of a variety of chemically different
macromolecules, such as polymeric proteins, teichoic acids, extracellular DNA and polysaccharides.
d. The pronounced recalcitrance of S. aureus joint infections to antibiotic treatment is due to
exceptionally strong bacterial aggregation and biofilm formation, which renders even high
concentrations of antibiotics given to patients ineffective.
METHODS, RESULTS, AND CONCLUSIONS
4. How was the research conducted and with what materials and methods (experimentally or
otherwise), and why were these methods used?
a. The research team started by draining human synovial fluid (SF) from the joint during total knee
arthroplasty in the operating room and collected this with permission of the Thomas Jefferson
University Institutional

The Health Of The Livestock Production Of Poultry Carrying...
The Acute phase proteins are produced as the develop of a response caused after a variety of
conditions stimulated by inflammatory cells producing chemical substances that activate specific
organs to produce these proteins to reconstitute homeostasis and thus restore the injured tissue. Even
though the Acute Phase Response generated is not specific for each determined disease, the
measurement of these proteins in an animal may indicate the occurrence of an unhealthy condition,
and a further diagnosis and prognosis of the illness could be accomplish. Regarding veterinary
public health, the livestock production represents a significant field from where products for human
consumption are provided. Thus, the measurement of these proteins in ... Show more content on
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These are plasma proteins which are released into the blood stream, mainly produced in the liver by
the hepatocytes (Cray et al., 2009) as part of the Acute Phase Response (APR), that either increase
or decrease in response to the tissue damage (Jain et al., 2011; Stahl, 1987; Gruys et al., 2005). The
APR is a non–specific and complex systemic reaction, part of the early–defense or innate immune
system, which is triggered by different stimuli such as infections, trauma and inflammations in order
to reconstitute homeostasis and induce healing (Cray et al., 2009; Tothova et al., 2014). As well as in
humans, APP in animals are sensitive to similar triggering events in response to inflammatory
stimuli. A variety of APP have been recorded, such as: fibrinogen, haptoglobin (HP), serum amyloid
A (SAA); C–reactive protein (CRP); ceruloplasmin (CER), major acute phase protein (MAP) and α1
acid glycoprotein (AGP) (Stahl, 1987; Gruys et al., 2005); differentiating their levels according to
each species (Cray et al., 2009; Eckersall and Bell, 2010). Thus, APP have been described for their
application in human medicine to diagnose and prognosticate different inflammatory and organ
diseases. However; altered levels of APP monitored in animals might provide an alternative method
to measure conditions or situations that may be influencing the concentration of these proteins in
plasma (Tothova et al., 2014). For instance, in livestock; APP have been presented as markers of
health

Subject Identification And Enrollment For Alzheimer 's...
Subject identification and enrollment Subjects recruited for this study consisted of individuals aged
60 to 85, with 10 or more years of education or more. Those diagnosed with Alzheimer's disease
(AD) and mild cognitive impairment (MCI) were recruited to the study at the Memory Clinic of the
Jewish General Hospital (JGH) in Montréal, and normal community elderly volunteers were
recruited through advertising. Subjects received standardized clinical, mental status,
neuropsychological, and cognitive evaluation. Depression was excluded via the Yesavage Geriatric
Depression Scale (Geriatric Depression scale > 15) [37]. None had evidence of cerebral infarct on
CT, and all subjects scored less than 4 on the Hachinski ischemic scale [38]. No individuals had
evidence of significant heart disease, alcoholism, or drug use and all showed normal routine blood
work. Subjects were classified as normal if they lacked subjective memory complaints, and scored at
or above 26 on the Montreal Cognitive Assessment test [39], and scored above 25 on the Folstein
Mini–mental state exam (MMSE) [40]. Their average MoCA score was 27 (range 25–29). The AD
patients received the diagnosis of mild, moderate, or severe probable AD according to standard
clinical criteria [41]. They had mild, moderate, or severe probable AD [42], scored below 26 on the
Montreal Cognitive Assessment test [39], scored between 4 and 25 on the Folstein Mini–mental
state exam (MMSE) [40], and showed

The Cardiovascular System And Its Blood Transporters Essay
Introduction: The cardiovascular system and its blood transporters continue to amaze scientists, as
wells, as others due to its fascinating functions. The cardiovascular system inhales oxygen and
functions to pump blood. It also, transports nutrients throughout the body. This system, then,
administers a crucial chemical reaction between the blood and hemoglobin. Hemoglobin is a
particular protein responsible for binding to oxygen molecules and transporting those molecules to
the body's tissues. Because of hemoglobin's important function, the body is able to properly
distribute oxygen molecules, proteins, and hormones throughout the whole body.
A spectrophotometer becomes a useful tool in determining the concentration and the identity of an
unknown sample. The data gathered from a spectroscope measures absorbance through light waves.
A spectrometer measures how much a sample absorbs light as light passes through the sample inside
of the device. Also, spectronomy is a quantitative analysis, which absorbs light over a variety of
wavelengths. It depicts the movement of electrons. More concentrated solutions will possess a
higher absorbance due to the electrons being excited and moving into higher energy levels. The
principle of spectrometry can be applied when determining the concentration of an unknown sample
as well. From the information gathered, one can create a standard curve in order to discover the
range of absorbance and the identity of the unknown sample.
A standard

Bradford Assay On Unknown Concentrations Of Proteins
Bradford Assay on Unknown Concentrations of Proteins
Taylor Coleman
September 27, 2016
Lab Group 3
BIOL 1111: General Biology Lab
Fall 2015
Section 107
Chad Perry
Abstract Colorimetric assay is a process of determining a concentration of a solution based on
absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way
to determine an unknown concentration of two samples of protein. The Bradford assay is done by
measuring wavelength of light passing through a cuvette filled with Bradford dye and
concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a
spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard
curve based on concentration (x–axis) and wavelength (y–axis). The standard curve is then used to
measure an educated guess on the concentrations of unknown protein concentrations. We
hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can
determine an accurate concentration of two unknown concentrations of proteins. The results of this
lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The
standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to
demine the concentration of an unknown concentration.
Introduction Colorimetric assay is a process determining the concentration of a chemical element or
compound in a solution

Microcapsules Essay
Layer–by–Layer Approach for Making Microcapsules
Muhammad Faiz Omar (201308264)
4th December 2014
Abstract
The fabrication of hollow multilayer microcapsules in the beginning is initiated by layer–by–layer
(LbL) self–assembly technique of oppositely charged polyelectrolyte on colloidal template followed
by core decomposition. The main concept of microencapsulation is to protect the contents of
fabricated microcapsules from the harmful environment in which it is surrounded. In this literature
review, I shall outline the technique of microcapsules encapsulation employing LbL approach, lists
of polyelectrolytes and templates and application of microcapsules.
Introduction
Over the past decades, nanometre–sized or known as microcapsules have been benefited the
approach of Layer–by–Layer(LbL) technique due to the ability of tailoring their properties(e.g., size
, shape, colloidal stability, surface–functionality, composition) that are biocompatible and
biodegradable.1–3,10They are broadly used in many applications including food technologies,
cosmetics, textiles industry and pharmaceuticals. The LBL assembly of multilayers are
fundamentally built up by the alternating deposition of oppositely charged polyelectrolytes onto a
sacrificial template such that the charge oscillates between positive and negative with each layer.1,4
The establishment of nanocoatings using LbL assembly method distinguishes itself in its
intelligibility compared to the more conventional method. The

Hi-Res Standard Agae Dyes
2.3.0 DNA Quality For each individual DNA sample 2µl of DNA, 8µl of deionised water and 2µl of
6x blue/orange loading dye (ThermoFisher) was added to a microcentrifuge tube and mixed using
the pipet. The DNA solutions were then refrigerated while a 2% agarose gel was made. Initially 2g
of Hi–Res standard agarose (Bioproducts Ltd. Yorkshire, UK) was weighted out on a balance
(Denver Instrument S–203) and added to a 250ml conical flask. Once this was completed 98ml of
deionised water was added and heated on a hot plate (Halogen Hotplate–HT2). The flask was
continuously heated and stirred until the agarose powder had fully dissolved. Then the solution was
cooled by running the base of the conical flask under water while stirring. Once the solution had
begun to cool 2ml of 50X Tris–acetate–EDTA (TAE) (ThermoFisher) and 10µl of Gel Red Nucleic
Acid Stain 10,000X in DMSO (Biotium. Fremont, USA) via pipetting. Once mixed the solution was
poured into gel electrophoreses tank including well comb and left to set for 30 minutes. Once set the
gel is immerged in approximately 300ml of 1x TAE, then 10µl of DNA solutions including a PCR
sizer 100bp DNA ladder (Norgen Biotek Corporation. Thorold, Canada) ... Show more content on
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Buckinghamshire, UK) were used within each supplied tube. Each bead contains the desired
ingredients needed for the successful PCR reaction, stabilizers, bovine serum albumin to increase
overall yield, four deoxynucleotides triphosphates (dNPT), adenine, guanine, cytosine, tyramine
ATP, approximately 2.5 units of puReTaq DNA polymerase and a reaction buffer. When
reconstituted to the final volume of 22µl, the final volume of each dNTP is 176µM in 8.8mM of
Tris–HCL which at room temperature has a pH of 9.0. Finally, 44mM of potassium chloride and
0.66mM of magnesium chloride. All PCR was preformed within a fume cupboard (Bassaire 06HB)
to minimise the risk of either moisture or genomic

Physioex 12 Answers
Review Sheet
Exercise 12
Serological Testing
NAME Kali Rothfuss
LAB DATE/TIME 6/22/11
Positive and Negative Controls
1. Why are there a number of washing steps in serological tests? The are a number of steps needed
in order to remove any non specific binding that may have occurred.
2. Describe how you would know that you had a "false positive" result. What does this mean for the
rest of your results?
A positive result with a negative control indicates a "false positive" and your results are invalidated.
3. Describe how you would know that you had a "false negative" result. What does this mean for the
rest of your results?
A negative results with a positive control indicates a "false negative" and your ... Show more content
on Helpwriting.net ...
19. If a patient tests positive for HIV using the ELISA test but negative using Western blotting, what
can you say about the initial ELISA result?
The initial ELISA result was a false positive but the Western blot confirmed this positive as false.
20. Describe the role of the developing buffer in the Western blotting technique. The developing
buffer contains the secondary antibody that the enzyme alkaline phosphatase are conjugated to.
21. Which patient(s) tested positive for HIV using the Western blotting technique? Patient C tested
positive for HIV
22. Using the choices listed below, decide which of the criteria apply to the indirect ELISA, which
apply to the Western blotting technique, and which apply to both. Use "E" if it applies to ELISA,
"W" for Western blotting, and "B" for both.
Criteria:

E 1. It tests for the presence of antibodies in the patient sample.
W 2. Electrophoresis is used.
E 3. Substrates that change from colorless to colored are used.
B 4. An enzyme–linked secondary antibody is used.
E 5. Antibodies against individual antigens are detected.
E 6. The reaction takes place in a well in a microtiter plate.
E 7. An antigen–antibody reaction is detected.
E 8. An optical density is read to determine whether or not a sample is

Enzyme Linked Immunisirbent Assay
The major use of Enzyme Linked Immunosorbent Assay (ELISA) is to titrate antibodies against
specific antigens (Crowther, 1995). This method involves an enzyme, which reacts with a colourless
substrate to form a coloured product, is covalently linked to a specific antibody that recognises a
specific antigen. If the antigen is present, the antibody–enzyme complex will bind to it, and the
enzyme component of the antibody–enzyme complex will catalyse the reaction which results in a
coloured product (Berg et al, 2002). The presence of the coloured product indicates that an antigen
is present. The ELISA, which is rapid and convenient, can detect less than a nanogram (10–9 g) of a
protein and can be performed with either polyclonal antibodies, which are antibodies that can
recognise multiple epitopes on any one antigen or monoclonal antibodies, which are antibodies that
can only detect one epitope on the antigen (Guo et al, 2006). However, the use of monoclonal
antibodies gives more reliable and accurate results due to their specificity as monoclonal antibodies
are very effective at acting as the primary antibody in an assay, or for detecting antigens within a
tissue. Also, they will usually result in reduced background staining compared to that of polyclonal
antibodies.
Indirect ELISA and direct ELISA Different types of ELISA's have been developed with
modifications to the basic steps. The key step with the ELISA assay is the direct or indirect detection
of antigen by

Reaction Of The Maillard Reaction
Introduction
The non–enzymatic reaction of reducing sugars with amino groups of amino acids, peptides and
proteins is called as non–enzymatic glycosylation or simply glycation. This non–enzymatic reaction
ultimately results in the formation of complex brown pigments and protein–protein crosslinks that
were first studied under defined conditions by Maillard in the early 1900s (Maillard and Gautier,
1912) and the reaction came to known as the Maillard reaction. There is a condensation reaction
between reducing sugar and free amino acids at the amino terminus or susceptible ε–amino group of
lysine residues of proteins producing labile N–substituted glycosylamine (Schiff base) that may
undergo two sequential rearrangements, forming a stable Amadori product (Fayle & Gerrard, 2002).
The reducing sugars namely, glucose, fructose, galactose, mannose, ribose and certain carbohydrate
relatives are inherently reactive toward nucleophilic groups. Glucose is the least reactive among
common sugars because it exists in least concentration in the open chain form (Paulsen and
Pflughaupt, 1980). Amadori rearrangement was a key early step in the Maillard reaction (Hodge,
1955). Early glycation directs the formation of Schiff's bases and Amadori products that further
oxidized to advanced glycated end products (AGEs) (Chetyrkin et al., 2008). Studies have shown
that the glycation process (in the early stage where the interaction of sugars, such as glucose, with
free amino groups of lysine and

Analysis Lab Report Sample
Table 1 OD of E.coli Cell Cultures Compared to Blank Samples 7 Table 2 The Averages Absorbance
of Bovine Serum Albumin In E.coli Induction Experiment 7 Table 3 The Averages Absorbance of
The Cell Lysate & Cell Pellet In E.coli Induction Experiment 7 Table 4 The Concentration of The
Cell Lysate & Cell Pellet In E.coli Induction Experiment 8 Table 5 Calculation of SDS Standard
Curve for E.coli Induction Experiment 16 Table 6 The Calculated Mw of PTEN in E.coli Induction
Experiment 8 Table 7: The Averages Absorbance of All Samples Proceed Along With Affinity
Chromatography Experiment 16 Table 8 The Calculated Concentration of Affinity Chromatography
Experiment 9 Table 9 The Calculation of SDS–PAGE Standard Curve for Affinity Chromatography
Experiment 17 Table 10 The Mw of PTEN In Affinity Chromatography Experiment 10 Table 11 The
Calculation of Mammalian Cell Density 10 ... Show more content on Helpwriting.net ...
Table of Figures Figure 1 Order of The Samples Loaded to SDS–PAGE for E.coli Induction
Experiment 8 Figure 2 SDS–PAGE for E.coli Induction Experiment 8 Figure 3 The Samples Order
of Affinity Chromotography Expermint 9 Figure 4 SDS– PAGE of Affinity Chromotography
Expermint 9 Figure 5 The Order of Western Blotting Samples 11 Figure 6 The PVDF Membrane
Showing The ladder after Westren Blotting 11 Figure 7 The Chemiluminscence Reaction on PVDF
Membrane 11 Table of Graphs Graph 1 Bradford Assay Standard Curve for E.coli Induction 7 Graph
2 SDS Standard Curve for E.coli Induction Experiment 16 Graph 3 Affinity Chromatography of
His6 Fused PTEN 9 Graph 4 The SDS–PAGE Standard Curve for Affinity Chromatography
Experiment

Ligand Bonding Lab Report
For this experiment, ligand binding was introduce into this course. What is ligand binding? It is a
smaller molecule that bind to a larger molecule, called a substrate (Berger). Bovine serum albumin
(BSA) is a protein that derive from cow that has a large molecular weight up to 66,436 Da. Phenol
red dye has a molecular weight of 354.88 Da, and is binding to bovine serum albumin at pH 4, pH 6,
pH 8, and pH 10, and our target was to determine the optimal pH. The optimal pH ranging between
3 to 5, and that it will appear to have two peaks on the graph. The first peak would be the phenol
red–BSA at maximum and the second peak is only phenol red at maximum because it does not bind
to BSA. After gather each pH peaks, then calculate the percent bound.

Should Vaccinations Be A Choice?
Should Vaccinations Be a Choice? According to scientists, a vaccines dominant purpose is to
customize immune systems and to allow them to work in contradiction of diverse viruses and
infections. However, in place of assisting the struggle against infection and certain diseases,
vaccines can be the key cause of infections and illness (Sepkowitz, 2012). Therefore, vaccinations
should be an individual choice and not a requirement by state and school legislation. The first
vaccination invented was for smallpox in 1798 by a man named, Edward Jenner (Kotz, 2009). His
work was herald as the leading scientific effort to control a transmittable virus by the measured
consumption of a vaccination. Today, current medication is founded on Pasteur's germ philosophy of
infection (Link, 2005). Scientists now have vaccinations for almost everything from vitamin
vaccinations to vaccines preventing illnesses. Oklahoma school and state legislation requires a
variety of vaccinations and require them at various ages from as early as three months through
eighteen years of age. Some of these vaccinations come with severe side effects if any side effects at
all. Also these vaccinations come with a possibility of causing the infection that the vaccine was
made to prevent. This is when the situation of an individual's immune system comes into play
whether to accept or reject the vaccination. Vaccinations that are required by the state and school
legislation of Oklahoma are; DTaP (diphtheria,

The Specificity of Albumin Binding Experiment
The specificity of albumin binding experiment was to determine the binding interactions that occur
between serum albumin and three synthetic dyes with the use of electrophoretic procedure. Whole
blood, or plasma. Clots upon standing and if the clot is removed, the remaining straw colored fluid
is called serum. The major protein in serum is albumin which functions as a carrier molecule for the
transport of certain small molecular weight compounds in blood. Molecules that bind to serum
albumin are fatty acids, hormones and some synthetic dyes. In this experiment the synthetic dyes
used are Bromophenol Blue, Ponceau S and Orange G. we observed that free dyes not bound to
albumin migrate faster that albumin or dyes bound to albumin. This ... Show more content on
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The 2nd half for the gel is our experiment compared to Anderson group 6. The 1st layer in all 3
pictures is the bound dye, the 2nd layer is the free dye.
Fig1 shows on the right group one next to it, 1st half of our result on electrophroresis gel. The 2nd
half for the gel is our experiment compared to Anderson group 6. The 1st layer in all 3 pictures is the
bound dye, the 2nd layer is the free dye.
Group 1, the gel picture and Andersons experiment are closely related. The binding did occur, in the
1st lane of group 1the dye was free therefore no binding occurred because no Bovine Serum
Albumin (BSA) was present in the mixture. While in lanes 2 to 4 binding did occur because there
was BSA in the mixture, with the higest binding occurring in lane 4, this is because there was no
buffer to interrupt the binding. (saturation).
Group 6, which is the 2nd half of the gel picture, does not correspond with Anderson results, no
visible binding occurred. This group had two different dyes and SDS included in the mixture also. In
Andersons version although two different dyes were used, it didn't affect his results, it's the SDS that

Gfba Gene Essay
Effect of cultivation conditions and gfba gene on formation of biofilm by Streptococcus dysgalactiae
Abstract
The pathogenesis of Streptococcus dysgalactiae is attributed to a combination of extracellular
factors and properties such as adherence and biofilm formation. The aim of this work was to
evaluate the influence of different factors, additives and bovine milk compounds on S. dysgalactiae
biofilm formation, as the presence of the gfba gene by PCR. Additionally, extracellular DNA and the
effect of DNaseI were evaluated in the biofilms yielded. Optimal biofilm development was observed
when the pH was adjusted to 7.0 at 37 ºC. Additives as glucose and lactose reduced biofilm
formation as bovine milk compounds tested. PCR assay showed ... Show more content on
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Treatment of the disease is currently based on antimicrobial susceptibility tests according to Clinical
and Laboratory Standards Institute (CLSI) [5]. Standard therapy designed for bacteria isolates at
planktonic state continues to be applied. However, the pathogenic profile and the antimicrobial
resistance are totally different from biofilms, causing reduced cure rates. Furthermore, the ability to
form biofilm is important both from the pathogenicity to the animal as for the manufacturing milk
industries, where the pathogen can adhere to abiotic food processing structures and persist in
adverse conditions through biofilms. The development and establishment of the biofilm depend on
the ability of pathogen to adhere to bovine mammary epithelial cells. S. dysgalactiae have many
virulence factors including the capacity to bind to the host's cells surface by a protein called GFBA,
which is involved in adherence and internalization, by its linking to lactoferrin [6]. It was
demonstrated that the S. dysgalactiae isolated from mastitis cows was able to produce biofilm [7].
Economic negative effects in the milk production around the world and the possible involvement of
biofilms in S. dysgalactiae mastitis infections, address the research to the study of factors that may
contribute for the biofilm production in order to establish more effective treatment strategies.
Different studies report that environmental conditions influence the capacity to form biofilm in

What Are Gel Electrophoresis?
Gel electrophoresis is a gel technique that separates DNA and proteins based on their mass, by
means of an applied electrical field that passes through an agarose or polyacrylamide gel. The
concentration of agarose in the gel is commonly 0.8% to 1.0%, since agarose is expensive. The gel
is embedded on a buffer, pH of 8.3, which keeps the pH of the solution at equilibrium. Assuming
that at typical pH, DNA is negatively charged, denatured protein samples are placed in the wells
located on the negative electrode side; hence the positive electrode side is located at the other end of
the gel. As the positive and negative electrode sides are connected to a power source, protein sample
migrates to the positive side of the gel. Migration of proteins is not necessarily based on their mass
or mass to charge ratio; protein migration across the gel is based on their size. In other words
smaller molecules will travels further than bigger molecules. Since the SDS gel contains agarose or
polyacrilamide, molecules have to be small enough to migrate to the other end of the gel without
getting stuck on the way. Protein samples separated by size via SDS–Page can be identified using
mass spectroscopy; which will require proteins from the gel to be treated using trypsin; which is an
enzyme that digests proteins. Assuming that samples were treated and storaged properly, the
digestion of peptides begins with the rehydration of proteins using trypsin, followed by the
incubation of proteins using a

Stem Cells And Its Effects On The Field Of Bioengineering
Stem cells has outgrown its medico–therapies in the field of bioengineering and also in the treatment
of various conditions of the blood and immune system, or to cure specific cancers Since it's a known
fact that stem cells can different to specialized cells in an undifferentiated manner. As the paper
states the use of insulin producing cells from human or rat adipose tissue –derived stem cells
transduced with pancreatic duodenal homeobox can could cure type 1 diabetes. An alternative is, use
of embryonic stem cells. Embryonic stem cells display the ability to differentiate in vitro into variety
of cell lineages. Insulin–secreting cells are derived from the mouse embryonic stem cells which can
normalize blood glucose when transplanted ... Show more content on Helpwriting.net ...
An alternative treatment to type 2 diabetes is insulin therapy since it is difficult to achieve desired
glucose levels due to increased spread of the disease. Transplantation of pancreas or replacement of
patient's islets of Langerhans is an ongoing treatment capable of achieving normoglycemia.
However the limitations of immunesupression and scarcity of donor pancreas make this procedure
not very desirable.
Stem cells therapy has been considerd as an alternative stratergy for replacement of damaged –cells ,
example embryonic stem cells , bone marrow stem cells etc. Initially to cure type 1 diabetes ,
insulin–producing cells were created by transducing bone marrow stem cells with the pancreatic
duodenal homobox (Pdx1) gene. Although this experiment was successful in normalizing glucose
levels in streptozotocin–induced diabetic mice, the low level of insulin production and the
unlikeliness of producing the culture conditions in vivo make this approach unfit for clinical
applications. Moreover, there was only a modest improvement in blood glucose levels by using
experiments done by western blot, immunofluorscence and RT–PCR. We need to further tune this
treatment strategy to improve blood glucose levels to be able to beat the insulin therapy. A very
efficient way to approach this problem is using insulin–secreting cells derived

Total Serum Protein Determination Lab Report
Introduction
Total serum protein determination is a standard clinical test that is often used for the diagnosis of
diseases involving the kidneys, liver or bone marrow and other metabolic or nutritional disorders1.
Blood plasma contains approximately 7% proteins by weight and the fluid that remains after the
clotting factors are removed from the plasma is the serum2. Albumin and globulin are the most
common blood proteins, and the total protein is the combined measure of the two3. The most
abundant protein is albumin, which acts as a carrier of many different molecules in the blood and
helps maintain blood volume2. The globulin proteins include many enzymes and elements of the
immune system such as antibodies and immunoglobulins2. Changes in ... Show more content on
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This method is a chromogenic test that uses biuret reagent to detect the presence of peptide bonds in
blood serum12. The active ingredients in the reagent are sodium hydroxide, potassium sodium
tartrate and copper sulphate pentahydrate13. The development of a violet colour after incubation at
room temperature is indicative of the presence of proteins and peptides in the blood serum12. A
coloured coordination complex forms when the copper ions (Cu2+) binds to the deprotonated
nitrogen atoms that form the peptide bonds of the amino acids12. Reactions can also occur between
Cu2+ and the nitrogen in the amide groups and the imidazole rings12. The colour intensifies after a
period of incubation as the alkalinity of the solution increases and more deprotonated nitrogen atoms
become available12. The concentration of the protein and the colour intensity are proportional,
thereby exhibiting a linear relationship in accordance with the Beer–Lambert law14. Absorbance is
measured by spectrophotometer, λmax = 540

Cell Cycle Analysis Paper
Cell cycle analysis. PC3 (8×104 cells/well) and LNCaP (3×105 cells/well) cells were seeded in 6–
well plates in 1.5 mL of complete growth media. Cells were treated with 2.5 μM of free sorafenib or
an equivalent sorafenib concentration of SMA–Sor, 3 μM of free nilotinib or an equivalent nilotinib
concentration of SMA–Nilo, DMSO or SMA for 48 h. Cell cycle distribution was assessed using
propidium iodide staining, as previously described {Somers–Edgar, 2011 #82}. Samples were
analysed using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the
proportion of cells in each of G0/G1–, S– and G2/M–phases were determined using CellQuest Pro
software (BD Biosciences, San Jose, CA, USA). Apoptosis analysis. PC3 (8×104 cells/well) ...
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The cells were washed with PBS and then incubated in serum–free media and treated with 2.5 μM
of free sorafenib or an equivalent sorafenib concentration of SMA–Sor, 3 μM of free nilotinib or an
equivalent nilotinib concentration of SMA–Nilo, DMSO or SMA for 48 h. Following treatment,
media was collected, centrifuged to remove cell debris, and freeze–dried for 12 h. Samples were
rehydrated and mixed with loading buffer (0.4 M Tris, pH 6.8, 5% SDS, 20% glycerol, 0.03%
bromophenol blue). For zymography, samples were loaded on a 10% SDS–polyacrylamide gel
containing 1 mg/mL of gelatin. After electrophoresis, the gels were incubated in renaturing solution
(2.5% Triton–X–100 (w/v)) for 30 min at room temperature and then for 24 h at 37°C in a
developing buffer containing 50 mM Tris, pH 7.5, 200 mM NaCl, 4 mM CaCl2, and 0.02% NP40.
The gels were then stained with Coomassie blue R250, and regions without staining were indicative
of gelatin lysis. The gels were briefly rinsed and scanned. For MMP–9 and isthmin–1 secretion,
samples were loaded on a 10% SDS–polyacrylamide gel, and the expression of MMP–9 and
isthmin–1 assessed by western blotting using anti–MMP–9 (D6O3H, Cell Signaling) and anti–
isminth–1 antibodies (Biorbyt, San Francisco, CA,

Fluorescence Spectroscopy And The Emission Wavelength Of...
Abstract Fluorescent properties are used to study protein and small molecule interactions.
Fluorescent spectrophotometry determined the excitation wavelength of eosin isothiocyanate to be
525 nm and its emission wavelength was 545 nm. Glycogen phosphorylase was similarly studied.
The excitation wavelength was 330 nm and the emission wavelength was 360 nm. The emission
wavelength could be indicative of the presence of the fluorophore, tryptophan. The potential
interaction between bovine serum albumin (BSA) and 1–anilino–8–naphthalene sulfonic acid (ANS)
was studied through fluorescence and fluorescence resonance energy transfer (FRET). BSA had an
emission wavelength of 358 nm which could also be indicative of a tryptophan. When BSA and
ANS were mixed together, the emission wavelength was longer suggesting that the molecules
interact with each other and follow FRET.
Introduction
Fluorescence spectroscopy is a useful technique to determine the proteins and nucleic acids of a
macromolecule. Some molecules, fluorophores, can absorb light at certain wavelengths and then
emit it at another wavelength. When the molecule absorbs light it is excitation and the light released
is emission. The light particles, photons, hit a fluorophore to excite the molecule (Voet et. al 2013).
Excitation occurs by the valence electrons of the molecule receiving energy and moving to a higher
energy state from the ground state. The excited electrons will later return to the ground state because
they

Lab Report : Bca Protein Assay
Shrutika Mintri/ E2 Date : 09/17/2014 Lab Report #1 Project Title: BCA Protein Assay Purpose:
BCA (Bicinchoninic acid) Assay otherwise known as the "Smith Assay" has the fundamental
purpose of determining the protein concentration of the two given unknown proteins in the sample
solutions. The absorbance is measured using a Plate reader and a Standard curve is generated. Also,
the different types of pipetting techniques are assessed in this Assay. Methods: (1) There are two
methods using which the BCA Protein Assay can be performed– Test tube method and Micro plate
method. The following experiment has been performed using the Microplate procedure. Vial Volume
of Diluent Volume and Source of BSA Final BSA Concentration A 325 μL 325μL of Stock
1000μg/ml B 325μL 325μL of vial A dilution 500μg/ml C 325μL 325μL of vial B dilution 250μg/ml
D 325μL 325μL of vial C dilution 125μg/ml E 400μL 0 0μg/ml= Blank 1. Preparation of Diluted
Albumin (BSA) Standards: The following Diluted Albumin (BSA) Standards have been prepared
using Bovine Serum Albumin (BSA) Standard Solution (2.0mg/ml) and the Diluent Phosphate
Buffered Saline (PBS) having

Spectrophotometer: Concentration and Standard Curve Essay
Lab Report 4
Biology 103
Prof. Dr. Johnson
Spectrophotometer; the finding of protein concentration of an unknown sample of BSA, and by
using the standard curve.
Abstract A spectrophotometer's purpose is to use colors of the light spectrum to determine the
concentration of light absorbing molecules in a solution. (p.59) In this particular lab, our mission
was to determine the protein concentration and the standard curve of the unknown sample of BSA.
This, by preparing five dilutions of the unknown solution of BSA together with other known
concentrations, and then experimenting by observing how the concentrations were passed through
the spectrophotometer. The outcome resolved in the absorption levels being decreased, and this ...
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The same solution of 0.5 ml BSA was then added from test tube 1 to the test tube 2 after being
properly mixed, and from test tube 2 the solution was being added to test tube 3, and so forth all the
way up to test tube 5, with the same exact procedure. From the last tube, we then disposed the 0.5
ml solution. After above procedures, we now labeled another test tube "blank"; 0.5 ml blank distilled
water was purred into the tube with the serial dilution of 1:10. We also had a tube C labeled
"unknown" with the same 0.5 ml of solution. And after adding 5ml of Coomassie Blue to each tube
(1–5) and to the blank, the result of absorbance was read at 595 nm.
Result | Protein Concentration(µg/ml) | Absorbance(595 nm) | Tube 1 | 120 | .922 | Tube 2 | 60 | .557
| Tube 3 | 30 | .377 | Tube 4 | 15 | .180 | Tube 5 | 7.5 | .082 | Unknown: .759 |
The ending result of this experiment confirms that as five test tubes are lined up with the varying
level of absorbance, different results in the level of absorbance will appear as well, this is visible in
above table. Thus, this is due to the varying amount of water in the solution. The blank sample had a
0.30 in its level of absorbance.
Calculating serial dilutions and their concentrations
(Examples of test tube 1 and test tube 2for respective dilution and concentration)
Dilution or D = previous dilution (if any) ×the

The Criteria For American College Of Rheumatology ( Acr )...
Subjects
Thirty patients that fulfilled the criteria of American College of Rheumatology (ACR) for RA will
be included in our study. According to 2010 ACR Classification Criteria for RA equal or more than
6 criteria = definite RA
Thirteen healthy persons were also, included in our study as a standard group.
All persons were subjected to:
Full history taking include:
Age and sex.
Anti–Rheumatic drugs administration.
Underlying medical conditions: liver diseases, renal failure, malignant diseases, infectious diseases
or other inflammatory diseases.
Exclusion criteria:
1. Renal or hepatic disorders
2. Platelets < 150000 & WBCs < 3500
3. Other autoimmune disease
4. Malignancy
5. Cardiac diseases
Clinical examination:
All patients will be assessed according to 2010 ACR Classification Criteria for RA equal or more
than 6 criteria = definite RA.
Disease activity was assessed with the 28–joint disease activity score (DAS28).
Routine Laboratory investigation:
Complete blood picture was done for healthy and RA patient groups.
The erythrocyte sedimentation rate (ESR) and C–reactive protein (CRP) will be done.
Rheumatoid factor (RF)–IgM was determined in all patients by means of latex agglutination.
Serum levels of anti–cyclic citrullinated peptide (anti–CCP) were measured by ELISA.

Concerning the administered basic therapy, all patients received MTX dose 17.5 mg/week. In
addition, patients received hydroxychloroquine

The Infection Of Cattle And Dogs
The genus Babesia is composed of tick–borne intraerythrocytic apicomplexan protozoans that cause
babesiosis in mammals, including humans (Schnittger et al., 2012). The infection of cattle with
Babesia bovis causes massive economic losses in the livestock industry worldwide (Bock et al.,
2004; Malak et al., 2012).
Similarly, Babesia gibsoni infects dogs worldwide, causing serious concern among pet owners with
regard to animal health and welfare (Trotta et al., 2009; Yeagley et al., 2009). Cattle and dogs
clinically infected with B. bovis and B. gibsoni manifest fever, anemia jaundice, hemoglobinuria,
hypertensive shock, and more often deaths if left untreated (Bock et al., 2004). Due to the limited
range of anti–Babesia drugs available, the treatment of babesiosis is becoming increasingly difficult,
particularly in areas where the disease is endemic (Gohil et al., 2012; Lin et al., 2012). Therefore, it
is clear that alternative strategies are required for the discovery and development of the much
needed anti–Babesia drugs.
The M17 leucine aminopeptidases of Plasmodium falciparum
(PfM17LAP) and Plasmodium vivax (PvM17LAP) have been characterized and validated as a drug
target (Cunningham et al.,
2008; Lee et al., 2010). The malaria parasite, just like Babesia species, destroys erythrocytes,
causing many of the clinical symptoms responsible for the disease. The PfM17LAP is involved in
host cell hemoglobin digestion, metabolic regulation, intracellular housekeeping, and antigen

Separation Of The Proteins Involved
Separation of the proteins involved in both species blood and serum revealed that each was
composed of a major protein. A prominent protein in the rabbit blood was found accumulating at
approximately 13kDa, as shown in Figure 1 and Figure 2. It was concluded that this protein was
most likely hemoglobin, given the weight and large amount present in all blood due to its necessary
function of transporting oxygen and carbon dioxide throughout the body. Hemoglobin is a
"tetrameric protein of about 64.5 kDa, consisting of two –chains and two –chains" which breaks
down into each subunit having an approximate weight of 15kDa (Yu et al, 1997). This was in
correlation with the data present in Figure 1, given that the major protein present in rabbit blood was
approximately 13kDa, which would be the subunits that make up hemoglobin.
We hypothesized that similar proteins would also be present in bovine blood, yet the major band
present in this sample was located at much higher molecular weight of approximately 45 kDa.
Potential causes in weight differences could be the longer amino acid sequence present in rabbit
hemoglobin, 170 amino acids, versus the 145 amino acids present in bovine hemoglobin, yet this
would result in bovine being further down on the gel versus what is actually shown (Altschul et al,
1997). Given the data obtained, potential misidentification of hemoglobin appearing as the main
protein present or possible error in lane one (rabbit blood), could explain the anomaly in

The Spectrophotometer Determination of Protein...
The Spectrophotometer Determination Of Protein Concentrations And The Effects Sodium Dodecyl
Sulphate And Triton X–100 Have On Protein Concentration.
INTRODUCTION
Spectroscopy is used as a collective term for all the analytical techniques based on the interaction of
light and matter. Spectrophotometry is one of the branches of spectroscopy where we measure the
absorption of light by molecules that are in a gas or vapour state or dissolved molecules/ions
(Tombs, et.al, 1959). Spectroscopy is the use of electromagnetic radiation by a sample in order to
identify components of the sample (qualitative analysis) or to measure the amount of material in that
sample (quantitative analysis) (Tombs, et.al, 1959). Absorption is a process in which ... Show more
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MATERIALS AND METHODS
The BIORAD dye was firstly prepared by dissolving 0.225g of Coomassie Brilliant Blue G–250
(Merck, South Africa), in 67.5ml of Methanol (Riedel–de–Haёn, Germany) and 291ml of 85% (w/v)
Phosphoric Acid, diluted to 450ml with dH2O. Six test tubes were labelled SC1–6 and the 0.1mg/ml
stock of Bovine Serum Albumin (Roche Diagnostics, Germany) and dH2O was pipette into their
respective test tubes according to Table 1.
Table 1: Table of the BSA and dH2O volumes to be pipette and placed in the spectrophotometer to
obtain absorbencies for the standard curve
Test Tube Number Total Volume (μl) Protein (BSA) Stock (μl) H2O (μl) Amount of Protein (μg)
1 2400 0 2400 0
2 2400 50 2350 5
3 2400 100 2300 10
4 2400 150 2250 15
5 2400 200 2200 20
6 2400 250 2150 25
600μl of the BIORAD dye agent was pipette into each test tube making up the volume to 3000μl.
All the test tubes were then covered with Parafilm and placed in the Vortex (Lab Pro cc) for a few
seconds and they were incubated at room temperature for 5–60 minutes. Simultaneously another set
of test tubes were labelled X1–8 and pipette with 0.1mg/ml Lysozyme stock (Merck, Germany),
BSA, 1% (w/v) Sodium Dodecyl Sulphate prepared with dH2O, 1% (v/v)

Mandatory Synthesis Essay
Beef Allergies and Nutritional Alternatives on Those Affected There have been a significant number
of studies indicating a growing beef allergic response in our population (Fiocchi et al. 2000).
Moreover, a high incidence of atopic dermatitis among children and the elderly raises concerns
because of its direct impact with nutrition and well–being (Fiocchi et al. 2000). As a result, different
types of food challenges, test on the skin such as skin–prick test(SPT) and careful observation of
immediate whale–and–flare reactions have emerged (Fiocchi et al. 2000). Diagnosing Bovine Serum
Albumin(BSA), Ovine Serum Albumin(OSA) and Alpha–gal as the main anaphylaxis agents that
Immunoglobulin G (IgG) response is directed. Resulting in different nutritional ... Show more
Anaphylaxis responses are becoming more common due to genetic and environmental factors (Saleh
et al. 2012). BSA, OSA and Alpha–gal continue to be the main allergic agents known today
(McNeill and Van–Elswyk 2012). Health and well–being g hand–to–hand with a proper diet. As a
result of atopic dermatitis, different options have been adopted for proper intake of micronutrients
and essential vitamins of those affected (Asp et al. 2012). In specific, the children and elderly still
remain the most affected due to this condition (Saleh et al. 2012). Asp, M.L., Richardson, J.R.,
Collene, A.L., Droll, K.R. and Belury, M.A. Dietary protein and beef consumption predict for
markers of muscles mass and nutrition status in older adults. J.Nutr.Health.Aging. 16(9), 784–
790(2012).
Fiocchi, A., Restani, P. and Riva, E. Beef allergy in children. Nutrition. 16(6), 454–457(2000).
McNeill, S. and Van–Elswyk, M.E. Red meat in global nutrition. Meat.Scie. 92(3), 166–173(2012).
Saleh, H., Embry, S., Nauli, A., Atyia, S. and Krishnaswamy, G. Anaphylactic reactions to
oligosaccharides in red meat: a syndrome in evolution. Clin. Mol. Allergy. 10(5), 1–11(2012).
Van–Nunen, S. Tick–induced allergies: mammalian meat allergy, tick anaphylaxis and their
significance. Asia.Pac.Allergy. 5(1),

Membranes Case Study
GO has also been mixed with the polymeric matrix to fabricate different nanocomposite membranes,
such as polyvinylidene fluoride (PVDF) MF/UF [141,224,225], polyether sulfone (PES) NF
membranes [226] and PSf [162]. One of the main drawbacks of the blending technique is the
absence of GO direct exposure to foulants, which restricts the interaction between foulants and GO.
Accordingly, the superior GO features, in terms of antifouling, will not be entirely exploited. The
direct deposition of GO nanosheets on the membrane surface is a more effective technique for
fouling control, hence the GO/foulants interaction can be maximized. The exceptional structure of
GO confers a considerably greater contact area for the convenient interaction ... Show more content
on Helpwriting.net ...
The employment of GO leads to the production of small, uniform, stable and distributed AgNPs in
addition to improved silver loading and ion release control. Several applicable and effective
techniques for the fabrication of freestanding ultrathin rGO membranes were reported. One of these
techniques was exemplified by Liu et al [220], in which a reinforced GO membrane was shaped on a
mixed cellulose ester (MCE) filter through the filteration of diluted GO dispersion. The formed
GO/MCE membrane was then positioned over a hydriodic acid solution, where the GO membrane
rapidly changed to a black colour, confirming the reduction of GO to rGO by the acid vapour.
Furthermore, a new water filtration membrane was fabricated by LbL–SA as it is an effective
method to generate dense layers on a membrane support [229]. This method was successfully used
to integrate the negatively charged GO nanosheets with a porous poly(acrylonitrile) (PAN) support
through the positively charged poly (allylamine hydrochloride) (PAH) by electrostatic interaction
[211]. Additionally, GO–based FO membranes were fabricated in several studies. The first trial was
conducted by Park et al [222], in which GO nanosheets were combined with the PSf to produce a
novel membrane with a PSf/GO composite support layer. The PA active layer was then shaped on
the PSf/GO via IP to obtain the

Succinate Dehydrogenase Lab
Introduction
In this Succinate Dehydrogenase (SDH) project, we were expected to learn the process of cell
fractionation, the importance of the Bradford assay, how enzymes work what factors affect them,
how to adequately micropipette, how to generate a proper hypothesis and test it, and finally,
interpret our results and write a conclusion which answers the central question of this lab: how will
the assigned reagent affect SDH activity?
The SDH project was completed over a span of three weeks, where we had a primary concentration
each week that revolved around a core principal of this project. In week two, which was the first
week of this project, we blended and centrifuged our cell fractions in order to isolate them, and then
we used a sample ... Show more content on Helpwriting.net ...
absorbance. We graphed the results from the Bradford Assay on excel and created a linear standard
curve and equation for it, which was used to determine the protein concentration in our cellular
fractions. The data from the cellular fractions was used to discover the amount of specific SDH
activity in lysates. Furthermore, we also conducted the SDH assay in order to gain proper and
knowledgeable experience about how to assess SDH activity for our crude and mitochondrial
cellular fractions, and we also worked with enzymes to further learn about how they work and how
they are affected through various factors. The second week of this lab was rather heavy; it had many
components to it, each equally important for our end result and conclusion. The concentration in
week two was on the Bradford assay, which was imperative since it measured protein concentration,
and practicing with the SDH assay, which measured the activity of our crude and mitochondrial
fractions, and was therefore vital in the final week of the project.
During the final week of the project, we performed the SDH assay twice to test our hypothesis and
answer the primary question of this project: the impact of the assigned reagent (Malonate for us) on
SDH activity in our mitochondrial fraction. Week three's concentration was on correctly performing
the SDH assay twice in order to minimize human error, and to interpret our data for our lab

Glorimetric Blocking Solution Essay
 Coating solution (pH = 9.6): 1.59 g Na2CO3,2.93 g NaHCO3, add sterile deionized water
(ddH2O) and dilute to 1000 mL, autoclave spare .
 Washing buffer solution (pH = 7.4): 8.0 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 3.58 g Na2HPO4
12H2O, 0.5 mL Tween–20, plus ddH2O volume to 1000 mL.
 Blocking solution: 1 g of bovine serum albumin (BSA), plus washing liquid volume to 100 mL or
0.1 mol citric acid solution: 2.1 g of citric acid, plus ddH2O volume to 100 mL.
 Substrate buffer (pH = 5.0): 25.7 mL 0.2 mol / L Na2HPO4, 24.3 mL 0.1 mol / L citric acid
solution , 50 mL ddH2O.
 Stop Solution to 2 mol / L sulfuric acid: 22.2 mL of concentrated sulfuric acid, plus ddH2O
volume to 200 mL.
(1) The package was suppressed kisspeptin–54 antigen: each well of 100 ng / 100 μL with leaving
three empty holes blank. The plate was sealed using sealing plate incubated at 4 ° C ... Show more
An appropriate step has been accordance to kit instructions to determine the concentration range,
intra–assay precision and cross–reactivity with other related proteins.
Assay Procedure
1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and
Samples were added in duplicate to the Microtiter plate.
2. Add 50μl of Standard or Sample to the appropriate wells. Blank well did not receive anything.
3. Add 100μl of enzyme conjugate to standard wells and sample wells except for the blank well
cover with an adhesive strip and incubated for 60 minutes at 37°C.
4. Wash the Microtiter Plate 4 times.
Aspirate all wells, then wash plates four times using Wash Buffer (1X) and set fill volume at 350
μL/well/wash. After the final wash, invert plate and blot dry by hitting plate onto absorbent paper or
paper towels until no moisture

Assay For Quantification Of Anti Rabbit Proteins
9/30/2014
Rakshitha. P. Miskin
01399629
Tuesday– 10
Lab #2
MicroELISA assay for quantification of anti– rabbit protein antibodies in mouse before and after
immunization
Objective
Preparation of microELISA plates for the detection of antibodies in sera from pre–immunized
mouse against rabbit protein and comparing this to sera extracted at weeks 1 and 2 after
immunization and re–immunization of the mouse to check for increased titer of antibodies in the
sera
Materials and methods
Preparation of phosphate buffer saline (PBS): A tablet of PBS sigma #P4417 was added to 200 mL
of water maintaining pH between 7.2–7.4.
Preparation of PBS/BSA: 10mL of 1% bovine serum albumin (BSA) in PBS was prepared (0.1mg
BSA in 10mL PBS) and the solution ... Show more content on Helpwriting.net ...
The strips were incubated for 30 minutes at room temperature and then washed 3 times with PBS–
tween. To each of the wells, 100µL of peroxidase conjugated goat anti– mouse immunoglobulin was
added and incubated at room temperature for 1 hour. The strips were washed again with PBS– tween
and then with PBS. 100µL each of activated ABTS (100µl hydrogen peroxide was added to 1mL 2,
2'–azino–bis (3–ethylbenzothiazoline–6–sulphonic acid) (ABTS)) was added to each of the wells.
The strips were incubated for a few minutes and the absorbance was read at 405nm in a
microELISA plate reader.
Calculations
Dilutions for strip 1:
Well # Sera Dilution Volume of sera Volume of PBS–tween Total volume
1 – Negative control – 100µL 100 µL
2 – Negative control – 100 µL 100 µL
3 Pre–immunized sera 1:10 22 µL 198 µL 220 µL
4 Week 1 immunized sera 1:10 20 µL 180 µL 200 µL
5, 6 1:100 25 µL (of 1:10 stock) 225 µL 250 µL
7 Positive control 100 µL – 100 µL
8 Positive control 100 µL – 100 µL

Dilutions for strip 2:
Well # Sera Dilution Volume of sera Volume of PBS–tween Total volume
1 – Negative control – 100µL 100 µL
2 – Negative control – 100 µL 100 µL
3 Pre–immunized sera 1:10 22 µL 198 µL 220 µL
4 Week 2 immunized sera 1:10 (Stock 1) 15 µL 135 µL 150 µL
5, 1:100 (Stock 2) 15 µL (of stock 1) 135 µL

The Bacterial Culture And Growth
Results
Bacterial culture and Growth
Isolates were resuscitated from storage by culturing on glucose yeast extract agar and single
colonies of the isolates inoculated on glucose yeast extract broth incubated at 28oC in a rotatory
shaker at 180 rpm showed that growth saturation at 0.8 O.D was after 24 hr of incubation showed
that Rhodococcus species are aerobic
Biochemical Assay
Determination of concentration of crude extract was based on Bradford Coomassie brilliant blue
assay which forms a complex between the protein dye and protein in solution which causes a shift in
the maximum absorption of light that passes the dye from 465 to 595 nm (Bradford, 1976). The
amount of light that passes through the protein solution is ... Show more content on Helpwriting.net
...
Fig5: Standard curve diagram of the concentration of BSA measured at an absorbance of 595 nm.
The volume of each sample for restriction digest was calculated using the formula; C1V1 = C2V2
Where C1 = Initial Concentration V2 = Initial Volume C2 = Final Concentration V2 = Final Volume
Table4: Concentration of crude extracts
Isolates Concentration of crude extracts (µl)
A
1.0
B 1.7
C 1.4
D 0.7
E 0.8
F 1.5
G 1.0
H 0.8
I 0.7
J 0.4

K 1.5
L 0.9
M 1.2
N 1.1
O 1.0
P 0.8
Q 1.5
R 1.0
S 1.5
T 1.2
U 1.7
V 1.2
W 0.9
X 1.6
Y 1.1
Z 1.6
Electrophoresis Analysis
Agarose gel electrophoresis used for the separation of the fragments was based on the migration of
the negatively charged DNA to the positively charged anode. DNA has a uniform mass/charge ratio
therefore allowing the molecules to be separated by size within an agarose gel such that the distance
travelled is inversely proportional to its molecular weight. The rate of migration is controlled by the
following; size of DNA molecule, agarose concentration, DNA conformation, voltage applied
presence of ethidium bromide, type of agarose and electrophoresis buffer.
Figure 6 present

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