Production Of Crude Extract

Production of Crude Extract
Crude Extract
In order to produce crude extract, bovine tissue was obtained, precisely minced to exclude extra fat, and then blended with pH 7.2 phosphate buffer.
The purpose of blending the tissue with the buffer was to pulverize the cells, causing them to release their contents evenly–most importantly lactate
dehydrogenase (LDH)–into the solution. Since other cell components such as proteases which reduce LDH were also released from the lysed cells, the
slurry was kept on ice to minimize their kinetic activity. The solution was then centrifuged, creating a pellet made of cellular debris and leaving behind
the crude product in the supernatant. Using data from the enzyme assay, the crude extract revealed 155В±7 mg total protein, 4980В±80 units of total
activity, 32В±2 units/mg of specific activity, a 100% yield, and a purification factor of 1 fold (Table 1).
40% Supernatant
The purpose of the 40% ammonium sulfate fractionation step is to extract impurities with low solubility–such as lipids–from the solution. Because
ammonium sulfate salt is extremely soluble, it hydrates competitively against weakly soluble impurities causing them to precipitate out of the solution
as a pellet. Hence, LDH is left behind in the 40% supernatant. This step revealed a slight drop in total protein amount from 155В±7 to 124В±5 mg and
comparatively no change in total activity from 4980В±80 to 4900В±100 units (Table 1). This data agrees with the salting out theory which states that
since only weakly
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Protein Concentration Lab Report
Figure 3. Bradford Standard Curve using 10mg/ml BSA Protein. 10mg/ml BSA protein stock was serially diluted to cover a 5–2000Вµg/mL
concentration range. 50 ВµL of dilutions were added to 1.5mL Bradford Reagent, left at room temperature for 5minutes, and assayed at 595nm,
against a blank of 50 ВµL DI H20 and 1.5mL Bradford Reagent.1 ________________________________________ From the Bradford standard curve
equation, the protein concentration of the crude lysate was calculated to be 0.1402 Вµg/mL, and the protein concentration in Fractions 1, 2 and 3 was
found to be 11.5038 Вµg/mL, 11.5038 Вµg/mL, and 26.6553 Вµg/mL, respectively. Although Fraction 2 appeared to have the most intense yellow
coloring, Fraction 3 had the highest protein concentration.... Show more content on Helpwriting.net ...
The Stokes radius of a molecule is the effective radius a molecule has as it moves through a solution. Molecular weight of a molecule is sometimes
correlated with the Stokes radius of a molecule, and thus the elution trend in gel–filtration, but this is not without exceptions. Thus, Stokes radius is a
better predictor, because although two molecules may have the same molecular weight, a longer, more branched molecule will have a larger Stokes
radius than a smaller molecule.1 In the separation of blue dextran, cytochrome c, and potassium chromate, the molecular weight of the molecules were
proportionate to their molecular sizes. The largest molecule, blue dextran, eluted first, followed by cytochrome c, and then the smallest molecule
potassium chromate. Blue dextran has the largest molecular weight, and potassium chromate has the smallest. The results for elution order are in
accordance with what the theoretical, predicted results were. The efficiency of the separation was not as ideal as would be predicted on the basis of
molecular weights. The molecular weights of the molecules are 3 orders of magnitude different, and should produce well–resolved peaks.1
Experimental visual results show peak overlapping around 2mL and 3mL, as the colors were mixed at these elution volumes. This peak overlapping
may be attributed to peak broadening. Peak broadening has many causes, but of particular

Electrospinning Essay
Electrospinning is a convenient method for fabricating various nanofibrous scaffolds for biomedical applications. In electrospinning process, a
conductive collector device plays a critical role in determining the shape and the structure of the nanofibrous scaffold; however, the preparation of the
collector is often complex. In this study, a novel, flexible, and inexpensive approach based on pencil–on–paper method was developed for preparing
collectors used in electrospinning. Graphite is a conductive material widely used as a component in the pencil core. A series of collectors were
prepared through drawing different patterns on folded and unfolded paper using a pencil, followed by successfully fabricating two–dimensional (2D)
nanofibrous ... Show more content on Helpwriting.net ...
We believe that the method developed in this study would provide a convenient approach to fabricate complex collectors for producing ordered 2D or
3D nanofibrous structures.
2. Materials and methods
2.1. Electrospinning
The collector was fabricated using a common 5B pencil and A4 paper. Paper was tailored to suitable size, and different patterns were drawn using a
pencil. Subsequently, the paper was folded in different shapes as collectors. Poly (Оµ–caprolactone) (PCL, MW = 60,000) was dissolved in
dichloromethane/ethanol (v:v =3:1) to form a homogeneous solution with a concentration of 15 wt%, and polyvinyl pyrrolidone (PVP, MW =
1,300,000) was dissolved in ethanol at a concentration of 8 wt%. After that, the solution was transferred to a syringe fastened on an infusion pump.
The solution was pumped at a rate of 0.5 mL/h. 8 kV voltage was applied between the needle and the collecter placed 10 cm away to form an
electrospinning jet. The collectors and fibers were observed using a camera (Nikon, Japan) and scanning electron microscopy (SEM) (JSM–7011F,
Japan).
2.2. Cell experiment
Murine calvarial preosteoblasts (MC3T3–E1) were selected as the model cell to evaluate the cytotoxicity of the electrospun fibers. The nanofibrous
membranes and films after sterilization under UV radiation were cut into small pieces, placed in a cell culture plate, and soaked in the culture medium
(DMEM/F–12

The Effects Of A Putative Anti Cancer Drug On Hela Cells
Introduction
Background
According to the Office of National Statistics, in 2013, 29% of deaths in the UK were cancer related, making it the leading broad cause of death in the
population. In the United States, 1 in 4 deaths are cancer related, and it is estimated that in a lifetime, there is 44% chance of contracting an invasive
cancer if you are male, and a 38% chance if you are female (Siegel et al. 2014). So, although our knowledge and education into different cancer
mutagens such as UV radiation, chemical carcinogens and viruses (Alberts et al. 2002), as well as cancer physiology and treatments may have
improved, leading to a decrease in cancer related mortalities, there are still huge advances to be had in the field (CRUK 2013). ... Show more content on
Helpwriting.net ...
In another review paper in Nature, Masters (2000) described how cancer cell lines are usually from fast growing, undifferentiated cancers that are
easily transported and proliferated. HeLa cells may be differentiated cervical carcinoma cells but they attain their ability to proliferate uncontrollably
from the protein HPV–18, which, combined with their ability to divide indefinitely, due to their chromosomes not shortening on replication, mean that
they are the perfect cell line to grow in vitro (Watson 2010). Therefore, the HeLa model was used worldwide in research for not only cancers and
development of different chemotherapeutic drugs such as Cannabinoids (Guzman 2003), but also in the research of genomics and vaccines including
that for polio. It should also be noted, that the conditions in a laboratory lend themselves to a very minimal level of genetic drift and combined with
control measures that assure that new cells are used every few weeks, the HeLa cell line is actually very similar to that of the original group of cells
(Masters 2000). These characteristics mean that HeLa cells are an ideal cell line to use in this experiment.
In this experiment, analysis of cell viability will be achieved through an MTT assay. The salt 3–(4,5–dimethyl–2–thiazolyl)–2,5–diphenyltetrazolium
bromide (MTT) is often used in cancer drug screening (Vistica et al. 1991) as it is

Identifying An Organism With A Biological Lens Essay
Spectrophotometrically Identifying Alfalfa Sprouts' Chloroplasts and Lysosomes Isolated in a Sucrose Gradient
Introduction: Conceptualizing an organism with a biological lens involves understanding the organism's components and how their individual
functions characterize the overarching entity. For a body, this means understanding organs and tissues. For acell, this means classifying its organelles
and discerning their functions. Analysis of subcellular components has become a major focus in biochemical studies, and it relies heavily on the
fractionation of organelles. Fractioning and isolating organelles essentially enables a scientist to elucidate organelle functions, which, as mentioned
before, adds definition to the all–encompassing image of a cell. Modern studies on this topic tend to target organelles with somewhat ambiguous
functions, like anammoxosomes and melanosomes. By isolating these subcellular components and analyzing them, biologists have unearthed
ammoxosomes' role in filling a large part of the Earth's atmosphere with nitrogen gas (Neumann et. al, 2014). In addition to this, other biological
organizations have managed to sequester intact melanosomes from cells, separating them from other items of similar densities, and study the activity
of V–Type ATPase proteins while they are in a subcellular membrane (Pelkonen et. al, 2016). The general procedure for executing an experiment like
these involves lysing cells, centrifuging their components, and separating

Disadvantages Of Refractometer
INTRODUCTION
A refractometer is aneoptical device that takes advantage of the fact that light passing through a liquid bends or refracts. The refractometer is a tool. It
is used to help you select higher quality of foods containing more vitamins and minerals. They are also used to determine the concentration of
dissolved solids in a solution. Cooking oil, molasses, syrup, and other thick liquids require a refractometerqcalibrated to read 30–90 Brix.
Refractometer also is used in wine making. (Harrill, 1998) refractometer is also used to test to quality of fruits and vegetables.
A hydrometer is an instrument used to measure the density of a liquid as compared to that of water. Hydrometers usually consist of a calibrated glass
tube ending in ... Show more content on Helpwriting.net ...
Why?
Temperature is an essential parameter because the index of refraction is affected by temperature. The speed of light in a liquid will increase when the
temperature increases. For instance, according to Hanson (2006), when there is 1В°C increasing in temperature, many organic liquids the index of
refraction decreases by nearly %0.05
What are the advantages and disadvantages of both instruments in measuring total soluble solid content? Refractometer advantages: Д°nexpensive
instrument
Refractometer disadvantages: Tedious calibration No instrument protocol Operator dependent readings( limited accuracy) Long and difficult
thermosetting (water bath is required after each experiment)
Hydrometer advantages: Quick instrument Simple method Inexpensive instrument
Hydrometer disadvantages; Breakable glassware especially be careful when it is dropped into solution Difficult to clean and dry it needs to be sure
that it is cleaned well because not enough to clean can cause measurement mistake Operator dependent readings Large sample volume required
("Comparision of different measuring techniques for density and

Biology Case Study
Materials and methods
The levels of microRNA–22–3p (miR–22–3p) and TP63 were analyzed by RT–qPCR and/or Western blot. Cell proliferation was measured using MTT
assay. Cell invasion and migration were evaluated using Transwell assay chambers. Prediction of a regulatory relationship between microRNA–22–3p
and 3'–UTR of TP63 mRNA was performed by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay.
OC cell lines
Lung cancer cell lines H292, PC–9, CL1–5, A549 and normal NHBE cells were used in this study. All of Lung cancer and normaL ceLL Lines– were
provided by ATCC. H292, PC–9, CL1–5, A549 and NHBE cells were cultured in RPMI–1640 medium supplemented with 10% fetal bovine serum
(FBS).
MiRNA and RNA interference ... Show more content on Helpwriting.net ...
The experiments were repeated thrice.
Cell migration and invasion assays
In vitro cell migration and invasion were examined using the Boyden Chamber Assays. For the cell migration assay, 30000 cells in 100 Ојl DMEM
medium devoid of fetal bovine serum (FBS) and a given concentration of cetuximab were cultured on a polycarbonate membrane insert coated with
fibronectin in a Boyden Chamber (Essen BioScience, UK). In the lower chamber, 500 Ојl DMEM with 10% FBS was added as chemoattractant. After
the cells were incubated for 6 h at 37 В°C in a 5% CO2 atmosphere, the insert was rinsed using PBS and cells on the upper side of the insert were
gently removed using a cotton swab. Subsequently, cells that adhered to the lower side were fixed with methanol, stained with crystal violet solution
and counted under a microscope in five predetermined fields (Г—200). All assays were independently repeated at least thrice. The same protocol as
described above was used for the cell invasion assay, except that the transwell filter membranes were pre–coated with 24 Ојg/Ојl matrigel (R&D
Systems, Inc., Minneapolis, MN, USA) and the cells were incubated for 8 h at 37 В°C in a 5% CO2 atmosphere.
RNA isolation, reverse transcription and RT–qPCR
RNA was extracted from the OC cells using the Trizol reagent. For miR–22–3p, RNA was transcribed into complementary DNA and amplified using
miRNA PrimeScript RT Enzyme Mix kit according to the manufacturer's instructions

Advantages And Disadvantages Of Cdna
troubleshooting. As the control RNA also did not yield any results through the several runs attempted, it is still unknown why long template cDNA was
not reliably produced for the next step of the sequencing process. It is possible further optimisation is required for cDNA to be synthesised, but funding
and time constraints limited it for this study.
Further studies in this area may use the same reagents and protocols used with further optimisation to yield results. Other possibilities are attempting
different primers to try and create cDNA that covers the whole genome.
4.7Conclusions
The aims of the study were to develop molecular assays to detect and type EV from patient samples and describe the virus distribution and
epidemiology in South Africa. A single EV real–time ... Show more content on Helpwriting.net ...
The advantage of the simplex, is that it is more robust and more sensitive that multiplex PCRs due to the nature of few primers and probes in the assay.
The disadvantage is that the sequencing assay to be based on these results needs to then amplify all EV, instead of more specifically a species per
assay. Nix et al developed an assay for this purpose in 2006 (Nix et al., 2006). This assay was able to detect all EV serotypes spread across all 4 HEV
species. In this study, more recently classified EV were detected by this assay: EV99, EV102, and EV114. These are little described viruses with no
clear disease association. The epidemiology of EV in South Africa showed a general concordance with other studies (Benschop et al., 2010, Apostol et
al., 2012, Dhole et al., 2009). HEV–B and HEV–C contained the most number of typed viruses in the country, though as mentioned, many previous
studies were biased due to the use of culture before virus typing, instead of typing directly from patient samples. Echovirus 30 is seen to be the
predominant serotype in Europe, but elsewhere in the world, the viral distribution is more even, with serotypes co–circulating (Oberste

Essay about Molecular Biology and Ldh
*Running title: Isolation and Characterization of Avian Lactate Dehydrogenase
To whom correspondence should be addressed Sylvia DaoudKinze and James Proestes, Department of Biochemistry, Portland State University
Professor, Portland Oregon, 97207–0751;
E–mail: sylviakinzie@gmail.com and proestoj@gmail.com
–––––––––––––––––––––––––––––––––––––––––––––––––
Keywords: Lactate, Dehydrogenase, Avian, Bradford Assay, Affinity Column
Background: Lactate Dehydrogenase also known as LDH is an important NADH dependent enzyme in metabolism that catalyzes the conversion of
Pyruvate to Lactate.
Results: Catalytic activity was detected from chicken breast muscle
Conclusion: Copious amount of catalytic activity was detected, indicating that ... Show more content on Helpwriting.net ...
Then the total of 51.32 grams of chicken was weighed out, cut into small pieces and placed in a blender along with 75 mL of cold extraction buffer.
Four short bursts (around 5 seconds per burst) were applied to complete the homogenization process with a ten second pause between each pulse. The
homogenized tissue solution was then poured into three 50 mL centrifuge tubes, and was centrifuged at 15000 RPM for 20 minutes. The supernatant
poured through two layers of cheese cloth into a 50 mL falcon tube, and the volume was recorded. Three 0.5 mL aliquots were saved.
Ammonium Sulfate Precipitation After centrifugation, the volume of each CFE was measured, and then per every mL, .39 grams of ammonium
sulfate was weighed out. The CFE was then placed in a beaker that was suspended on top of ice in a larger beaker, which was placed on top of a stir
plate. Over a course of 20 minutes, ammonium sulfate was added to the CFE while stirring slowly. After the completing adding all of the ammonium
sulfate, solution was stirred for an addition 15 minutes. Followed by centrifugation at 15000 RPM for 20 minutes.
Purification of LDH
Dialysis of the Protien Dialysis of the salted protein was completed by a lab T.A.
Affinity Column Chromatography To separate LDH from the rest of the protein, affinity column chromatography method was used. A column with a

plastic stopcock was loaded with Cibacron blue affinity matrix by adding

Resistance Of The Mutant Oncogenes
Introduction
One in five deaths today is a result of some form of cancer. This terrible disease stems from mutations in body cells, which interfere with the cells'
normal regulation of growth, division, and death. Cells can make occasional mistakes and divide too early or not destroy themselves at the right time
(Alberts et al. 2014). The Hayflick limit, described in 1961 by Leonard Hayflick, states that normal human cells can divide for about 50 times (Watts
2011). In cancers, though, this limit does not exist, and cells continually divide. Occasional mistakes do not have tremendous consequences on large,
multicellular organisms, however. When many cells begin to make these mistakes, though, tumorigenesis andcancer can occur. ... Show more content
on Helpwriting.net ...
The pigment protects skin cells, particularly the DNA of the cells, from damaging ultraviolet radiation from the sun, and has been shown to reduce the
frequency of radiation–induced mutations and to completely remove low–dose radiation effects (Mosse et al. 2000). Within melanocytes are
melanosomes, organelles in which melanin production occurs. The melanosomes have 4 stages of maturity, starting with stage 1, or premelanosomes,
and progressing to stage 4, or mature melanosomes. The stages can be determined based on the composition of the vesicles in the cells (Setaluri 2003).
The mature melanosomes then branch off from the dendritic tips of the melanocytes and travel to keratinocytes, keratin–producing cells (Seiberg 2001).
The chemotherapy drug methotrexate is an alkaloid that has been used as a treatment for acute lymphocytic leukemia, non–Hodgkin's lymphoma,
osteosarcoma, choriocarcinoma, head and neck cancer, and breast cancer. Methotrexate is an antimetabolite that acts as a folic acid antagonist.
Components of folate vitamins are required for DNA synthesis and cell replication, so by inhibiting folic acid and other folates, methotrexate slows the
DNA synthesis and division of certain cancer cells, and impacts cell proliferation (Jolviet et al. 1983). The estimated half maximal effective
concentration (EC50) for methotrexate was found to be 0.00339ОјM for human breast cancer MDA–MB–231

Advantages And Disadvantages Of Reverse Sequence Syphilis...
Introduction
The traditional algorithm for syphilis screening has involved use of a manual non–treponemal test (e.g. rapid plasma reagin, RPR) followed by
confirmation with a treponemal test (e.g. Treponema pallidum particle agglutination, TP–PA). Non–treponemal tests are inexpensive, but require
significant hands–on time by laboratory personnel and produce subjective results. High volume laboratories are increasingly using a reverse sequence
algorithm, using partially or fully automated treponemal tests (e.g. enzyme immunoassay–EIA, chemiluminescene immunoassay–CIA, multibead
immunoassay–MBIA), followed by a non–treponemal test (e.g. RPR, VDRL) if reactive (1). Potential advantages of the reverse sequence algorithm
include reduced labor ... Show more content on Helpwriting.net ...
Our objective was to determine signal strength cutoffs associated with 98%, 99%, and 100% reactive confirmatory testing (TP–PA or fluorescent
treponemal antibody absorbed test, FTA–ABS). For the four automated immunoassays we also attempted to establish a signal strength cutoff associated
with a consensus of at least 4/7 reactive treponemal tests (the index test plus at least 3 others). The findings from this study will inform laboratory
guidelines on use of confirmatory treponemal testing for patients with initially discordant serology.
Results
Among 1995 specimens tested, 907 (45%) were categorized as true positives and 1087(54%) as true negatives using a consensus of the test panel as
the gold standard (see Materials and Methods for case definitions). Table 1 includes results for positive agreement, negative agreement and Kappa's
statistics for the seven assays. Positive agreement was lowest for FTA–ABS (86.1%, 83.7–88.2%) and highest for Centaur EIA (99.7%, 99.0–99.9%).
Positive agreement

Clinical Signs Of Bvdv Infection
Laboratory diagnosis The clinical signs of BVDV infection are highly variable and there are many factors that affect severity of infection like viral
strain, age of affected animals, immune status, reproductive stage and mixed infection. Therefore, diagnosis based on history, clinical signs and
postmortem examination should be considered only as preliminary diagnosis. The accurate and confirmatory diagnosis of BVDV infection depends
only on laboratory diagnosis. 1– Virus isolation BVDV isolation in cell culture, followed by identification, is considered as the most reliable method
for diagnosis. Many different clinical samples are suitable for BVDV isolation during the viremia stage as nasal discharges, peripheral blood
leukocytes, serum, semen, aborted fetuses and feces. However, presence of maternal antibodies in serum and buffy coat samples from newly born
animals may neutralize the virus and decrease the sensitivity of virus isolation. Virus isolation methods are labor intensive and may take several
weeks. Furthermore, virus isolation cannot differentiate between PI animals and transiently infected animals. The FBS used as supplement for
propagation of cell lines should be tested for BVDV contamination before usage to avoid false positive results. Dubovi et al. 2013 stated that there are
many factors that may affect the success in BVDV isolation and discussed the misconception that BVDV isolation is the gold standard for diagnosis.
Some of those factors are variation

Enzyme Catalysis And Enzyme Inhibition Essay
Enzyme catalysis and enzyme inhibition are two essential biological mechanisms of organisms. In this experiment, WT–AP and MBP–AP enzyme are
reacted with different concentrations of PNPP substrate in SpectrovVis time based assays. From the change in absorbance over time data, and the rates
of the reactions are calculated, followed by the determination of the kinetic constants. Then, the MBP–AP assays are repeated with two different
concentrations of phosphate inhibitor and the kinetic constants of the uninhibited enzymes are compared to those of the inhibited enzymes. Results of
the experiment show that the uninhibited WT–AP and MBP–AP enzymes yielded expected kinetic constants according to a reference. However, the
inhibited enzymes produced kinetics constants that did not resemble a competitive inhibition, which was expected. Instead, the inhibitor is observed to
be a mixed inhibitor.
Introduction Enzymes are biological catalysts. Without them, biological reactions will not proceed in a reasonable rate. For example, without a series
of digestive enzymes, it can take 20 years to digest a hamburger. Enzyme–catalyzed reactions proceed through an ES complex, in which the substrate
binds to the enzyme, gets transformed into the product, and releases into the environment. For a specific enzyme, only one or a few different substrate
molecules can bind in the proper manner and produce a functional ES complex. The substrate must have a size, shape, and polarity compatible with

A Diagnostic Tool For Renal Disease And Hypertensive...
Introduction Proteinuria is often used to detect the presence of any type of proteins in the urine and serves as a diagnostic tool for renal disease and
hypertensive disorders. Among those disorders, pre–eclampsia is characterized by low renal perfusion and leakage of proteins in urine due to a
damaged glomerular base membrane (1). This condition is associated with a high risk of maternal complications and prenatal mortality. Pre–eclampsia
is usually defined as a combination of hypertension, edema and proteinuria after 20 weeks of gestation and is determined by a threshold of 300mg/24
hours of proteins (2). Normal urine excretion is up to 150mg/day (3). The most common test used in antenatal care to screen proteinuria is the visual
reagent dipstick. It is a semi–quantitative and colorimetric method. The stick changes colour when the reagent (tetrabromophenol) binds to the
proteins. The binding is pH dependent (albumin binds at a pH between 5 and 7, other proteins bind only at a pH below). If the urinalysis by the dipstick
is positive, a 24 hour–urine collection is performed in order to confirm the proteinuria and to estimate the total amount of protein (4). Another method
available for detecting proteinuria is called the Bradford assay. This quantitative method is based on the binding of proteins with the dye Coomassie
Blue which increases the absorption of the dye at 595nm. The absorbance measured by spectrophotometry is related to the amount of proteins present
in the

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

Questions On Materials And Methods
2– Materials and methods 1. Snails B. alexandrina snails (8 – 10 mm) from Medical Malacology Laboratory, Theodor Bilharz Research Institute
(TBRI), Giza, Egypt were used. Pieces of polyethylene sheets were put into the aquaria to collect egg masses. 2. Synthetic insecticide: Match 5% EC:
Lufenuron 5% EC (Registration No., 609) was purchased from Syngenta (AGRO–Egypt). 3. Bioassay tests: 3.1. Molluscicidal screening: A stock
solution of 1000 ppm was prepared from the insecticide on the basis of W/V using dechlorinated tap water. To calculate LC50 and LC90, Series of
concentrations were prepared and ten B. alexandrina snails were placed in beakers for each concentration (WHO, 1983) and another group in
dechlorinated water as control. Three replicates were prepared for each group and control, then, snails' mortality was recorded. 3.1.1. Collection of
hemolymph: To collect the hemolymph, a small portion of the shell which situated directly above the heart of snails was removed and a capillary tube
was inserted into the heart (Nduka and Harrison, 1980). a– Total hemocytes count: The number of cells was counted using a BГјrker– Turk
hemocytometer (Van der knap et al., 1981) by using 10 Вµl of hemolymph of each group. b– Differential hemocytes count: Hemocytes monolayers
were prepared according to the method of Abdul–Salam & Michelson (1980). c– Biochemical Assays: Activities of aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) in

Bradford And Lowry Assay Lab Report
The Bradford and Lowry assay are two color changing proteins with similar processes. Color changes of the solution can be measured using a device
called a spectrometer. In lab, the two assays can be used to determine the amount of protein in a solution. This can be done in an experiment by
reacting a specific reagent with amino acids or peptide bonds present in the proteins. This produces a color change in the solution depending on the
amount of peptide or amino acid reactions with the reagents. This color change can then be measured using a device called a spectrophotometer.
The Bradford assay is a one– step process, which involves the Coomassie dye reacting with the amino acids arginine and lysine. It under goes
changings in acidic conditions ... Show more content on Helpwriting.net ...
The type of amino acid sample affected the ability of the reagents to react with the peptide bonds and amino acids. It also affected the amount of amino
acids and peptide bonds in the protein. Each of the four proteins used in the experiment had the same concentration, which was reflected in the accuracy
of the standards. In relation to the Bradford reagent, the amount of reactions that occurred depended on the amount of arginine and lysine amino acids
present in the protein. In relation to the Lowry reagent, the amount of reactions that occurred depended on the amount of tryptophan and tyrosine
amino acids in the protein and the number of peptide bonds linking the amino acids. The absorbance measured in the spectrophotometer was affected
also by the differences in amino acid compositions and the ability of the reagents to react as a result. Since all the protein standards had the same
concentration of 0.5 mg/mL, it allowed for the accurate comparison of the absorbance, mean, and standard deviation of each of the

Disadvantages Of Fenugreek Seeds
Fenugreek seeds were bought from a local herbalist. The seeds were ground up and five different solvents were used: chloroform, 70% ethanol,
ethyl acetate, hexane and water. For each solvent, 2.5 grams of coarsely powdered seed material was extracted by refluxing with 25 ml of each
solvent stated above for a period of thirty minutes and kept overnight at room temperature before filtration. The following day, after filtration, the
solvents were evaporated until dryness using a rotary evaporator and the crude extracts were weighed. To make a stock solution that could be used for
the subsequent experiments, five millimeters of dimethyl sulfoxide (DMSO) was added and two hundred milligrams of the crude extract were
weighed. Throughout the entire experiment, the percentage of DMSO was kept constant (1%). MCF7 breast cancer cells were acquired from the
American Type Culture Collection (ATCCВ® HTB–22в„ў). The media was supplemented with 10 % (v/v) heat–inactivated fetal bovine serum (FBS)
(Gibco Invitrogen), antibiotics 100 U/ml penicillin (Lonza) and 100 Вµg/ml streptomycin (Lonza), 2 mM L–glutamine (Lonza) in addition to 25ВµM
4–(2–hydroxyethyl)–1 piperazineethanesulfonic acid (HEPES) (Biowest, France). Cells were maintained at 37 В°C, 5% CO2 humidified incubator.
The cell viability assay was performed using the 3–(4,5–dimethyl–2–thiazoyl)–2,5–diphenyl–2H–tetrazolium bromide (MTT) as described previously
(Mosmann, 1983), with minor modification. In summary, 7Г—103 cells were seeded

Lab Report On Luciferase
Introduction
In this experiment, the enzyme Luciferase (Luc) was purified using differential centrifugation, precipitation, and a simplified chromatography
procedure, followed by an enzyme activity assay using the enzyme's natural substrate, Luciferin, to confirm the presence of Luc.
Luciferase is a 61 kD enzyme naturally found in the tails of fireflies, a member of the Lampyridae family. Luc acts as a catalyst in an
oxidation–reduction reaction with Luciferin, which allows these insects to exhibit bioluminescence for mating and communication ("Firefly Luciferase
," n.d.). In this reaction, Luciferin is adenylated and oxidized to produce Oxyluciferin, along with carbon dioxide, adenosine monophosphate, and light
("Luciferase Mechanism," ... Show more content on Helpwriting.net ...
Following the purification process, the enzyme activity assay should reveal luminescence in the supernatant fraction of the tail sample. SDS–PAGE of
the samples should reveal a band at 61 kD in the tail sample and no distinct banding in the head sample.
Materials and Methods
Protein Purification:
To begin, samples of both firefly heads and firefly tails were divided amongst the groups. Our group was assigned the firefly head to use as a
negative control, while the other was assigned to the tail. The firefly tissue was lysed by grinding with a pestle in the tube (Tube A) both before and
after adding 250 L of Lysis buffer (1X PBS with 1% SDS). This mixture was then sonicated for two minutes to complete the lysing process.
The tubes were then centrifuged at 1,000 rpm for 3 minutes to remove the large tissue pieces. The supernatant of this tube contained the soluble
compounds and proteins that were present in the cytosol of the cells present in the tissue. The supernatant was removed using a micropipette and
transferred to a new 1.5 mL–Eppendorf tube (Tube B), and the pellet Tube A was discarded.
The supernatant in Tube B was then mixed with 1 mL of ice cold acetone in order to separate the proteins from the other compounds present in the
aqueous solution. Tube B was spun in the centrifuge at 10,000 rpm for 2 minutes. The resulting supernatant was removed and discarded to isolate the
pellet, containing the

Benefits Of Prunus Mume Fruit
Basic functions of prunus mume fruit inside the body
пѓјThe Citric acid present in the plum fruit helps in the absorption of vitamins and enzymes, thus preventing several diseases and reduces fatigue.
пѓјEating plum fruit on a regular basis can stimulate the secretion of parotid gland hormone, which can prevent aging process in humans.
пѓјPrunus mume intake increases metabolism and aids in the process of body's energy restoration functions in a natural manner
How the prunus mume fruit should be used
пѓјAlthough the Chinese Plum is edible, it should be taken with proper care for the desired effects. Below are some of the points to keep in mind
while consuming this fruit–
пѓјThe plum fruit should not be consumed by anyone suffering from cold or ... Show more content on Helpwriting.net ...
Additionally, they help break down the harmful buildup of toxic fats in the body system and aids in the process of purification of the body.
Benefits of Brown Rice Vinegar for the body
Among the several benefits of Brown Rice Vinegar for human body, the major ones are–
Slower the process of Ageing: The authentic brown rice vinegar contains approximately 20 amino acids and 16 organic acids that help prevent the
formation of toxic fat peroxides which generally contributes to the process of aging.
Maintains the cholesterol level for a healthy heart: Since it inhibits fatty peroxides, brown rice vinegar is known to help in the process of reducing or
preventing the buildup of harmful cholesterol on blood vessel walls thus helping in lowering the blood pressure, reducing the LDL cholesterol levels
and keeps the heart in better condition.
Improves the digestive health: The acetic acid contents of the brown rice vinegar helps in the absorption of essential vitamins and minerals in the body
thus improving the overall digestive health.
Basic functions of Brown Rice Vinegar inside the

Absorbance Values
Abstract:
Claims were made that manufacturers had improperly labeled protein concentrations on their products. Concentrations of these three different
protein–containing liquids, whole milk, soy milk, and a protein solution were determined using a Bradford dye reagent and a spectrophotometer to
measure the absorbance of each. Absorbance of a set of standard known protein concentrations from a range of 0.125 mg/ml to 2.000 mg/ml were
measured using the spectrophotometer at a set wavelength and this data was plotted. The equation generated from the best fit line of this data was used
to determine the unknown concentrations of the three different samples. These values were then compared to the protein concentrations obtained from
the corresponding ... Show more content on Helpwriting.net ...
This would indicate that either the food labels or the calculated data are incorrect. The data was dependent on a linear relationship between absorbance
and protein concentration. However, the linear relationship was based on only seven data points, two of which were omitted due to inconsistency, and
was only representative up to 2.000 mg/ml protein concentration. Because the data sample was small and covered a small range of concentrations, the
food label concentrations cannot be determined as inaccurate. The more likely cause would be that the linearity assumption of the correlation between
the absorbance and the protein concentration is

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 content on
Helpwriting.net ...
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–HaС‘n, 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 NumberTotal Volume (О
јl)Protein (BSA) Stock (Ојl)H2O (Ојl)Amount of Protein (Ојg)
12400024000
224005023505
32400100230010
42400150225015
52400200220020
62400250215025
600Ојl of the BIORAD dye agent was pipette into each test tube making up the volume to 3000Ојl. 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)

The Consequences Of Alcohol Analysis
Alcohol is a very widely used drug, and is therefore present in many clinical and forensic toxicology cases. Quantitation of alcohol levels in patients
affects the clinical treatment they receive as well as any legal consequences associated with the incident that led to their hospitalization. Alcohol
analysis and its consequences for postmortem cases also forms a large part of the forensic toxicologist's workload [1–2].
Although gas chromatography–mass spectrometry (GCMS) or gas chromatography flame ionization detection (GC–FID) is the most accurate and
reliable method of determining blood alcohol concentration (BAC), it is expensive, time–consuming, and requires technical proficiency manhours
[1–2]. Therefore, BAC determination in a ... Show more content on Helpwriting.net ...
However, there is still contention over its existence, prevalence, and severity; this is evidenced by recent articles and court cases contesting its
relevance [3,7] . It is problematic that almost every mention of this phenomenon goes back to a single study in 1995 that failed to put the issue to rest
[6].
Forensically, this research has multiple implications. The presence and level of alcohol in a victim or perpetrator's system has great bearing on their
behavior and frame of mind. As a result, the amount of alcohol in a person's blood can serve as a strong piece of evidence. From a sentencing
standpoint, the level of legal consequences assigned to an individual is also be dependent on the amount of intoxication detected [1]. With these
consequences in mind, it is imperative that the test used to measure ethanol levels is reliable and accurate. If there is a possibility that the enzymatic
assays used widely throughout hospitals can be inaccurate under certain conditions, it is necessary that the users and interpreters of those assays know
that interference can occur and when it might happen. If markers can be developed to determine when further testing for ethanol should be done, the
reliability of the test will be greatly increased. In this study, the aim will be to determine if lactate and LDH in clinically possible antemortem
concentrations can cause a significant

Bovine Blood Lab
For this experiment, whole bovine blood was used. The first process was to separate the blood into cellular and plasma fractions. 100 ВµL of whole
bovine blood was transferred into a yellow microcentrifuge tube that was labeled WB using a P–200. 50 ВµL of whole blood was added to a blue
microcentrifuge tube labeled WB. Both tubes were capped and placed in ice. 2 mL of the remaining blood was transferred into a Clear 2 mL tube
using a P–2000 and centrifuged for 5 minutes at 8000 RPM. Afterward, 800 ВµL of the supernatant from the Clear tube was transferred into a
yellow tube labeled WP and 50 ВµL of the supernatant was added to a blue tube labeled WP. These tubes were then capped and kept on ice.
Next, the plasma proteins from the blood were isolated ... Show more content on Helpwriting.net ...
A BSA standard serialdilution was created in wells A1–F1. Fifty microliters of dH20 were added to each well and fifty microliters of the BSA stock
was added to A1 and mixed. Fifty microliters from A1 was transferred to B1 and this was repeated for C1, D1, and E1. In wells A2–A4, triplicates
were created by transferring 10 microliters from A1. Triplicates were created for the other BSA standards using the same amount, but with a different
tip for each standard. The samples WP, WB, Es, and Ap (yellow tubes) were also used in the BradfordAssay. The dilutions for the samples were 1:10,
1:100, 1:500, and 1:1000. The first dilution consisted of 90 microliters of water and 10 microliters of the sample. The second dilution consisted of
90 microliters of water and 10 microliters of the sample as well. The third dilution composed of 80 microliters of water and 20 microliters of the
sample. The last dilution composed of 50 microliters of water and 50 microliters of the sample. These dilutions were used for each sample and
triplicates were generated for each dilution. Two hundred microliters were added to each triplicated well for all the samples, including the standard.
Then, the microplate was placed on the spectrophotometer and the absorbance was

The Effect Of Alkaline Phosphatase On Para...
A SPECTROPHOTOMETRIC ASSAY OF THE ACTIVITY OF ALKALINE PHOSPHATASE ON PARA
–NITROPHENYLPHOSPHATE IN THE
PRODUCTION OF PARA–NITROPHENOL
M. Smit1
1 Department of Biological Sciences, University of Cape Town, Rondebosch
Introduction:
Alkaline phosphatase is a biologically important enzyme, catalysing the hydrolysis of phosphate groups in organic compounds through the process of
dephosphorylation. para–Nitrophenylphosphate (pPNP) is an example of a chromogenic substrate of this enzyme, because the product of hydrolysis of
pPNP is the yellow para–nitrophenol (pNP) compound along with inorganic phosphate (McComb, Bowers and Posen, 1979).
The Michaelis–Menten equation for enzyme kinetics (Murray, 2002) predicts an initially high rate of enzyme activity as the concentration of the
substrate is relatively high, followed by a gradual reduction in enzyme activity as the substrate is consumed. The enzyme alkaline phosphatase is also
known to be subject to competitive inhibition by inorganic phosphates via the formation of the phospho–enzyme, thus blocking the active site to pPNP
for dephosphorylation (Kim and Wyckoff, 1991).
The enzyme kinetics of alkaline phosphatase on this chemogenic substrate is the focus of this study, in an attempt to determine the trend in the rate at
which pNP concentration changes over time and the impact of a high concentration of the inorganic phosphate product on enzyme activity. It is
hypothesised that the rate of enzyme activity will initially be

Potential Therapeutics For Treating Type 2 Diabetes Mellitus
Bromophenols have been identified as potential therapeutics for treating type 2 diabetes mellitus (T2DM) due to their suspected tyrosine phosphatase
1B (PTP1B) inhibitory activity. A new series of bromophenol analogues will be synthesised and their PTP1B inhibitory activity will be tested in in
vitro enzymatic assays to elucidate their mechanism of action as PTP1B inhibitors.
Background and Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic disease that is caused by insulin resistance of the peripheral tissues and impaired insulin secretion of
the pancreatic ОІ cells.1 Both conditions result in reduced glycemic control, which can lead to hyperglycemia. Despite the availability of a wide range
of anti–diabetic drugs to treat T2DM, ... Show more content on Helpwriting.net ...
Bromophenols have attracted much attention as potential PTP1B inhibitors due to their suspected anti–diabetic activity.4 Bromophenols occur
naturally in marine organisms such as algae, mollusks and jellyfish. (В±)–Polysiphenol (1, figure 1) is an example of a bromophenol that can be
extracted from the marine algae Polisiphonia Ferulecea.5 After the Braddock group successfully achieved the first total synthesis of (В±)–polysiphenol
and the separation of its enantiomers, they collaborated with the Woscholski group to test their inhibitory activity in PTP1B enzymatic assays.6,7 Both
enantiomers of polysiphenol were essentially found to be identical in PTP1B inhibitory activity. Thus polysiphenol, as single enantiomers, cannot be
responsible for the observed inhibition. This implicates a chemical change of polysiphenol into a non–chiral form is likely to take place under the
conditions of the assay. One possibility is the formation of a para–quinone methide species (4, figure 1), which in turn can tautomerise into an
ortho–quinone methide species (5, figure 1). Thus there are various possible reactive species that might be responsible for the observed inhibition of
PTP1B. The formation of quinones highlights a red flag in drug discovery due to their highly reactive nature. Quinones are Michael acceptors that are
known to cause toxicity in vivo by covalently binding to nucleophilic sites on proteins and DNA.8 Consequently, quinones

LDH Purification lab Report
PURIFICATION OF LACTATE DEHYDROGENASE FROM CHICKEN MUSCLE TISSUE
Abstract
The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body.
Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from
chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and
purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein
mixture by affinity chromatography and its activity was studied by ... Show more content on Helpwriting.net ...
10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted. Protein
Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent–compatible formulation based on bicinchoninic acid (BCA) for the
colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from
0–2000 Вµg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions
from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at
370C and then the absorbance was measured at 562nm.
Results/Discussion The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of techniques
including homogenization, ammonium sulfate precipitation, dialysis, and affinity chromatography. Activity and Protein assay were used to track the
overall amount of LDH present in the samples. Crude Extraction: Chicken muscle tissue was homogenized in a blender with cold extraction buffer in
order to lyse cells, releasing LDH into

Lab Report : Bca Protein Assay
Shrutika Mintri/ E2Date : 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. VialVolume of Diluent Volume and Source of BSAFinal BSA Concentration
A325 ОјL325ОјL of Stock1000Ојg/ml B325ОјL325ОјL of vial A dilution500Ојg/ml C325ОјL325ОјL of vial B dilution250Ојg/ml D325ОјL325ОјL of
vial C dilution125Ојg/ml E400ОјL00Ојg/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

Curcuma Longa Essay
RESULTS AND DISCUSSION
Qualitative Analysis
Results of Phytochemical Evaluation for Curcuma Longa Extract
The results of phytochemical evaluation of Curcuma Longa extract summarized in table 2.
Table 2: Shows the results of phytochemical evaluation of Curcuma Longa extract.
1Alkaloids+
2Tannins+
3Glycosides+
4Saponins+
5Flavonoids+
6Terpenoids+
7Phenol+
Figure 3: Illustrates the results of phytochemical tests of Curcuma Longa extract.
The previous table 2 and figure 3 show the results of applied phytochemical tests for constituents of Curcuma Longa extract which were all positive. The
results indicate the presence of the active constituents of curcuminoids. It also indicates the presence of alkaloids, tannins, phenolic compounds,
terpenoids, saponins and flavonoids. These phytochemicals were also reported in ... Show more content on Helpwriting.net ...
No.Concentration Ојg/mlAbsorbance
1100.18
2200.23
3300.278
4400.33
5500.38
Concentration of Curcuma Longa extract in mcg/ml

Figure 5: Illustrates calibration curve of standard curcumin.
From the result in table 6 and figure 5 which illustrated the calibration curve of the UV spectrophotometer with working standard Curcuma Longa
extract and the r2 = 0.9999.
Curcumin in Gel Formulation
Table 7: Shows linearity of curcumin in the gel formulation.
No.Concentration Ојg/mlAbsorbance
1100.127
2200.148
3300.166
4400.185
5500.206
Concentration of Curcuma Longa extract in mcg/ml
Figure 6: Illustrates the curve of curcumin in the gel formulation. Assay Test of Curcumin Content in Gel Formula
Assay of curcumin in gel formula was calculated using the following formula
Assay of curcumin = (absorbance of sample / absorbance of standard Г— concentration of standard / concentration of sample) 100
= (0.127/0.180 Г— 10/10) 100 =

Material And Methods : Bm Mscs Cultivation
Material and Methods: BM–MSCs cultivation: BM–MSCs were obtained from Institute Pasture of Iran and were cultured in Low Glucose Dulbecco 's
modified Eagle 's medium (Low Glc–DMEM) (Gibco Invitrogen, Karlsruhe, Germany) with 10% FBS (Gibco Invitrogen, Karlsruhe, Germany) and 1%
penicillin and streptomycin (Sigma–Aldrich, Schnelldorf, Germany). Then, BM–MSCs were incubated in humified incubator at 37ЛљC and 5% CO2.
Cell's medium was replaced every two days. U266 cell line: Confirmed U266 cells, a human myeloma cell line, were purchased from Institute Pasture
of Iran. U266 cells were grown in RPMI–1640 (Gibco Invitrogen, Karlsruhe, Germany) with 10% FBS (Gibco Invitrogen, Karlsruhe, Germany) and
1% penicillin and streptomycin (Sigma–Aldrich, Schnelldorf, Germany) in T25 flask culture (Orange Scientific, Braine–l 'Alleud, Belgium). Then,
U266 cells incubated in humified incubator at 37ЛљC and 5% CO2. The medium was changed every day during all steps of experiments and Trypan
blue staining was used for viability analyzing. BM–MSCs derived C.M preparation: The BM–MSCs were cultured in T25 flask culture (Orange
Scientific, Braine–l 'Alleud, Belgium) with DMEM medium and incubated for reaching to 60–70% confluency. In the next step, DMEM medium was
replaced with RPMI–1640 medium without FBS and was incubated for 72 hours (hrs). After incubation; medium was collected, cells and debris were
removed by centrifugation. Supernatant was concentrated by 10 kDa MW cut–off ultrafiltration

Lab Report
4.2 Materials and Method
4.2.1 Materials
The size of the particle should be large enough to allow three proteins coupling together onto one particle. Therefore, DynalВ® MyOneTM 1–Ојm
carboxyl particle (InvitrogenВ®) and MerckВ® EM1–100/40 particle were used as the carrier particles. If positive results were seen, the work would
be expanded to particles in other sizes, materials and manufacturers, such as AllrunВ® PM3–050 or DynalВ® M270 particles.
The proteins chosen for coupling were mouse IgG, streptavidin and APC. They carry different functions and could give a good reflection of the
possible functional proteins used in future particle application. Mouse IgG (LampireВ®) stock solution was filtered with a 0.45Ојm filter before
diluting into ... Show more content on Helpwriting.net ...
For quality control purpose, the CS&T particles were also running daily before testing the samples to re–set the configuration. The machine was
serviced and maintained routinely and worked in good condition.
96–well U–bottom white polypropylene plate (GrenierВ®) was used for fluorophore loading assay and particle washing. The 1.5ml polypropylene
reaction tubes (GrenierВ®) were used for general particle treatment and protein coupling. Also, tube magnet and plate magnet were used in particle
separation. All the consumables were routinely used in Anteo and they were proven suitable for treating magnetic particles.
4.2.2 Method
The experiment in this chapter was divided into two parts. First, the particles were coupled with signal proteins individually at different coupling
concentration, and a titration curve was generated to give supportive information for later co–coupling experiments, including the gate setting and
spectrum compensation values. Second, the particles were co–coupled with three proteins at different protein coupling concentration level. Under
different concentration combination, each protein would show different signal response and it help to determine the optimal loading concentration
used in multifunctional particle construction.
The main particle used in this experiment was DynalВ® MyOneTM

The shape and magnitude of the UV spectra depends on the...
The shape and magnitude of the UV spectra depends on the composition of amino acid in each protein. Due to the aromatic amino acid residues in
the protein, the observed UV absorbance was mainly in the 240 nm to 340 nm region. In Figure 1 to 3, the maximal absorbance of each protein was
approximately at 280 nm. The difference in magnitudes of the peak observed was linked to the differences in the amino acid contents in each of the
proteins. The peak of lysozyme was greater than those of BSA and gelatin, because lysozyme has a greater number of tryptophan residues. Lysozyme
has six tryptophan residues, whereas BSA and gelatin has two and zero, respectively (Department of Chemistry, 2014). Lysozyme has three times more
tryptophan residues ... Show more content on Helpwriting.net ...
In order to examine the variation in sensitivity of lysozyme, gelatin, and BSA, the absorbance reading was obtained using the Bradford and BCA assay
Gelatin is observed to have a lower absorbance reading than lysozyme in only the Bradford assay, while in the BCA assay gelatin was observed to
have about the same absorbance as lysozyme. In Bradford assay, the color yield for proteins with higher content of tryptophan, tyrosine, or cysteine
residue, will be higher. The observed protein sensitivities did not correlates with the amino acid content in the Bradford assay. As presented in Table
I, the mole fractions of lysine and arginine in BSA were 10 mole percent and 4 mole percent, respectively. The sensitivity should have been higher in
BSA than in the Bradford assy. However, the observed sensitivities did correlates with the amino acid contents in the BCA assay. Bradford assay
would be ideal if an accurate estimation of a newly isolated protein using BSA as a standard. With the data obtain from the Bradford assay, it shows
that it was more sensitive and quicker, also it had less interference from substance that were possibly present in the protein solution, expect for the
interfering substance SDS.
The interference of RNA and ME in the Bradford assay was compensated by using the blank, because the difference between the addition of the protein
and the non–protein blank

DAPI Stain Living Cells
Introduction
The number of cells is closely regulated by the rate of cell division and the rate of cell death. When cells become unneeded, they activate an
intracellular death program and commit suicide. This process is often known as apoptosis. Cells die for numerous reasons. For instance, cell death
regulates cell numbers and can adjust the number of cells needed for a particular function. When a structure a cell forms is no longer needed, the cell
will die. Cells can become damaged or stressed and trigger an activation from within the cell to kill itself. Proteolytic enzymes called capases mediate
apoptosis by triggering cell death by cleaving proteins in the cytoplasm and nucleus. The triggering process is initiated by death signals by either
extracellular or intracellular signals. Response to signals coming from ... Show more content on Helpwriting.net ...
Specific protein location and morphology of organelles can be found using DAPI stains. The DAPI stain is classified as a nuclear stain that binds A–T
regions of DNA. DAPI significantly increases the fluorescent properties and can be identified as a blue fluorescence of DAPI when processed by
UV light. The only implications include using fixed cells. Using a Mitotracker Red CMXRos, you can stain living cells mitochondria. Requiring an
active mitochondrial membrane potential the mitochondria oxidizes the Mitotracker probe. Fluorescence property is maintained after fixation. An
ER tracker viable stain uses the bodipy fluorophore. The glibenclamide probe selectively binds sulphonylurea receptors of ATP sensitive K+
channels that are localized in the ER. Another viable stain used was the Lysotracker. Its probes are very selective for acidic compartments. Its probe
is retained in the lysosome via protonation of the probe. These different stains are identified as the mitotracker will be red, lysotracker is green and the
Hoescht stain will be

Purification of lactate Dehydrogenase
Purification of Lactate Dehydrogenase
Results and Discussion
Monitoring Assays Enzyme activity assay and Bradford dye binding protein assay was used.
Ammonium Sulfate Fractionation In order to isolate and purify Lactate Dehydrogenase (LDH), we first extracted LDH from bovine muscle tissue. We
minced 8 grams of meat and homogenized using blender. The homogenate was centrifuged and the pellet consisting of membranes, organelles,
cytoskeletal components and structural fragments was discarded. The crude extract was subjected to 40% (NH4)2SO4 fractionation. The purpose of
this is to remove molecules of lesser solubility than LDH like lipids, fats and low soluble proteins. For this, 9.24 g of was slowly added to crude
extract while stirring reaching final solution saturation of 40%. The mixture was then centrifuged and the pellet consisting the contaminants was
discarded. From table I, the data indicate that the yield for this was 88% with purification factor of 1.5. The total protein in 40% supernatant was 121
mg which decreased from 210 mg present in crude extract while retaining 88% of LDH suggesting some contaminant proteins were removed. Due to
some discrepancies in protein data for crude extract and 40% supernatant, we re–assayed these. Even though week 3 data may not be very reliable, the
crude extract assay from week 2 and 40% supernatant assay from week 3 gave the most appropriate data with least error. The 40% supernatant was
then subjected to 60%

Cell Viaability Lab Report
experiment. (5%) HEPG2 RВІ=0.8354 PANC–1 RВІ= 0.9734 PC–3 RВІ= 0.9482 For each value shown we can determine that there is a strong
correlation between drug concentration and % cell viability. This can tell us that as the drug concentration increases the % cell viability decreases.
This shows that the chemotherapeutic agent is having the desired effect. 4.What conclusions can you draw from the results of the experiment? (10%) I
can draw from these results that as the chemotherapeutic drug concentration is increased the cell viability decreases. In HEPG2 the viability at the
control was 100% but when the drug was added at concentration of 40Вµm the cell viability decreased to 2.62%. From these examples we can deduce
that the chemotherapeutic drug added is successful in its use and that it is targeting the cancerous cells. The drug damages the genes present inside the
cell line... Show more content on Helpwriting.net ...
It achieves this my measuring the mitochondrial enzyme activity and as such is used to determine the cytotoxicity of potential medical drugs. It
measures the reduction of yellow 3–(4,5–dimethythiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase.
The MTT starts as yellow but as it enters the cells and passes into the mitochondria where it is reduced and changes in colour to a dark purple
formazan product. The cells are then solubilised with an organic solvent and released, solubilised formazan reagent is measured using a spectrometer
(Cree, 2011). Some errors that may occur while using this assay are that uneven evaporation of culture fluid in the wells may cause erroneous results.
The MTT solution is stable for 6 months when stored at 0В°C but at 2–8В°C for more than 2 weeks may cause decomposition and erroneous results
may occur. Microbial contamination will contribute to cleavage of MTT and the formation of MTT formazan, causing erroneous results (Sigma Aldrich,

Use Of Small Volume Fluids On Scale Of The Field Of...
ABSTRACT
Microfluidics biochips are revolutionary devices in the field of clinical diagnostics, DNA analysis and molecular biology. Biochips involve various
fields of science and engineering i.e., physics, chemistry, biochemistry, nanotechnology, fabrication technology and computer science. It uses small
volume fluids on scale of micro to nano liter for automatically carrying out the reactions needed for some biochemical assays. In this report I have
covered basic overview of biochips, kinds of biochips, areas of research, existing problems and finally presented work done till now in area of sample
preparation specifically dilution and mixing Algorithms for Digital Microfluidics Biochips.
INTRODUCTION
Microfluidics biochips are emerging devices for analysis of biological assay, and have its potential application in the field of healthcare because of its
point–of–care diagnostic capability. These chips are of very small size typically of few centimeters [4]. These chips also have its application in
Biological computing it can be used as sample preparation primitive which reduces the operations time. DNA hybridization can be reduced to 4min
from 24hrs [2] by using it. Due to its small size and low cost it feasible to use in place of bulky convention diagnosis devices.
These biochips also used to monitor toxicity of air and water. It has many advantages such as micro liter fluids amount required for assay operation,
high speed analysis, less cost in

The Importance Of Animal Uses In Animals
Humans have used animals for food, transport, companionship and experimental research to develop medicine and investigate the structure and
function of the body. This dates back to 130 AD, where the commonly used animals were pigs, monkeys, and dogs (Vaumans, 2005). Dogs are the
species most frequently used in the animal models and are referred to as "default non–rodent" (Smith et al., 2002). Especially, in toxicology studies,
dogs are the preferred choice due to their similarities of human toxicity (Smith et al., 2002). Today, 75–100 million vertebrates are used per year
worldwide for research, teaching, and testing (Vaumans, 2005). In many countries it is mandatory to grade the level of discomfort and pain for the
laboratory animal. Animals suffering from discomfort and/or stress can have a change in their endocrinology and immunology results (Vaumans,
2005). By testing oxytocin concentrations in dogs can help facilitate the transition into new social settings and into new laboratory settings (Stallings,
2015). In addition, oxytocin plays a role in non–social observations such as learning, anxiety, feeding, and pain (Stallings, 2015). All laboratory animals
fall under the Animal Welfare Act, which promotes animal welfare of research and laboratory animals and promotes anti
–cruelty laws. Currently,
according to American Association for Laboratory Animal Science, (AALAS) animal welfare and assess laboratory animals' pain and distress is
conducted by a trained laboratory

Production Of Crude Extract

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