- The document describes a methodology for using spectrophotometry and the Bradford assay to determine the protein concentration of various food samples.
- Standard curves were constructed using bovine gamma globulin standards of known concentrations. Absorbance readings of diluted food samples were then used to calculate protein concentrations based on the standard curve.
- Calculated protein concentrations from the experiment differed from labeled values, possibly due to errors in dilution or pipetting techniques. The highest measured concentration did not match the expected highest protein food.
Determination of protein concentration by Bradford method.pptxVijay Hemmadi
Bradford uses Coomasie Blue which is a dye that binds specifically to proteins. It is very accurate and sensitive, compatible with most buffers, sugars, and chaotropic agents but high concentrations of detergent interfere in the assay
Spectrophotometric methods for determoination of Proteins Sabahat Ali
Different types of assay are present of determination of proteins
Bicinchoninic acid assay, Biuret protein assay, Lowry test assay, Bradford protein assay & Warburg Christion (A280/A260)
Chemical lab report analysis of food additives by hplc and uv-visAwad Albalwi
Aims:
• To determine the concentration of some additives in various beverages and an unknown
sample using High Performance Liquid Chromatography (HPLC).
• To use ultraviolet absorbance to measure the food additives caffeine and benzoic acid in
(non-diet) soft drinks, using Beer’s law and by solving simultaneous linear equations.
• To compare and contrast these two analytical methods as applied to the determination of
artificial sweetners.
format
Determination of protein concentration by Bradford method.pptxVijay Hemmadi
Bradford uses Coomasie Blue which is a dye that binds specifically to proteins. It is very accurate and sensitive, compatible with most buffers, sugars, and chaotropic agents but high concentrations of detergent interfere in the assay
Spectrophotometric methods for determoination of Proteins Sabahat Ali
Different types of assay are present of determination of proteins
Bicinchoninic acid assay, Biuret protein assay, Lowry test assay, Bradford protein assay & Warburg Christion (A280/A260)
Chemical lab report analysis of food additives by hplc and uv-visAwad Albalwi
Aims:
• To determine the concentration of some additives in various beverages and an unknown
sample using High Performance Liquid Chromatography (HPLC).
• To use ultraviolet absorbance to measure the food additives caffeine and benzoic acid in
(non-diet) soft drinks, using Beer’s law and by solving simultaneous linear equations.
• To compare and contrast these two analytical methods as applied to the determination of
artificial sweetners.
format
The present document provide the information about Methods of extraction of drugs from the biological matrix (protein precipitation method, liquid extraction)
the presentation contain ways used to estimate proteins, this presentation prepared by TONNYBITE, a student from KILIMANJARO CHRISTIAN MEDICAL UNIVERSITY COLLEGE, TANZANIA
Electrophoresis:
Electrophoresis is separation technique based on movement of charge particle in an electric field.
Movement of charge particles can be determined by following formula--
V= Eq/f
Where,
V= Velocity of the charged particle;
E= electric field of the molecule;
q= Net charge of the molecule; and
f= Frictional co-efficient of the molecule
Types of electrophoresis:
1. Agarose gel electrophoresis ;
2. Poly-acryl amide gel electrophoresis [PAGE];
3. Sodium do-decyl sulphate Poly- acrylamide gel electrophoresis [SDS-PAGE] ;
4. Two dimensional –Poly-acrylamide gel electrophoresis [2D-PAGE];
5. Pulse field gel electrophoresis [PFGE];
6. Capillary gel electrophoresis [CGE]; and
7. Disc electrophoresis for Protein.
Application of electrophoresis:
1. Estimation of the DNA molecule.[ Agarose , PAGE ]
2. Analysis of PCR product. [ Agarose ]
3. Separation of restricted genomic DNA and RNA. [Agarose and PAGE respectively]
4. Conformation of newly isolated DNA .[Agarose]
5. Separation of most small fragments of DNA. [PAGE]
6. In forensic science.[Agarose , PAGE, SDS-PAGE, 2D PAGE ,Capillary gel electrophoresis , PFGE]
8. In determining molecular wt. of protein.[SDS-PAGE].etc
Escozine for Pets™ has 4 major production steps.
1. Collection of Scorpions from the Scorpion Reservation. 2. Extraction of venom, purification and therapeutic dose preparation. 3. Polarization of extract and quality control of Polarization 4. Manufacturing, quality control, warehouse and shipment.
The present document provide the information about Methods of extraction of drugs from the biological matrix (protein precipitation method, liquid extraction)
the presentation contain ways used to estimate proteins, this presentation prepared by TONNYBITE, a student from KILIMANJARO CHRISTIAN MEDICAL UNIVERSITY COLLEGE, TANZANIA
Electrophoresis:
Electrophoresis is separation technique based on movement of charge particle in an electric field.
Movement of charge particles can be determined by following formula--
V= Eq/f
Where,
V= Velocity of the charged particle;
E= electric field of the molecule;
q= Net charge of the molecule; and
f= Frictional co-efficient of the molecule
Types of electrophoresis:
1. Agarose gel electrophoresis ;
2. Poly-acryl amide gel electrophoresis [PAGE];
3. Sodium do-decyl sulphate Poly- acrylamide gel electrophoresis [SDS-PAGE] ;
4. Two dimensional –Poly-acrylamide gel electrophoresis [2D-PAGE];
5. Pulse field gel electrophoresis [PFGE];
6. Capillary gel electrophoresis [CGE]; and
7. Disc electrophoresis for Protein.
Application of electrophoresis:
1. Estimation of the DNA molecule.[ Agarose , PAGE ]
2. Analysis of PCR product. [ Agarose ]
3. Separation of restricted genomic DNA and RNA. [Agarose and PAGE respectively]
4. Conformation of newly isolated DNA .[Agarose]
5. Separation of most small fragments of DNA. [PAGE]
6. In forensic science.[Agarose , PAGE, SDS-PAGE, 2D PAGE ,Capillary gel electrophoresis , PFGE]
8. In determining molecular wt. of protein.[SDS-PAGE].etc
Escozine for Pets™ has 4 major production steps.
1. Collection of Scorpions from the Scorpion Reservation. 2. Extraction of venom, purification and therapeutic dose preparation. 3. Polarization of extract and quality control of Polarization 4. Manufacturing, quality control, warehouse and shipment.
This simple laboratory PPT was designed for UPES-SOHST students as a guide for illustrating the experiment mentioned above, kindly share to help someone learn
Final submission –Pay attention to APA formatting, spelling, andChereCheek752
Final submission –
Pay attention to APA formatting, spelling, and grammar. Your similarity index/plagiarism score must be below 10%. Higher scores may impact your grade.
The final submission is the combination of the other four phases into one paper. You will combine Phase I, Phase II, Phase III, and Phase IV to make Phase V. You are responsible for editing and formatting your paper so that your paper will flow for the reader. This paper will need to be corrected with all the feedback provided from previous papers. Include conclusion and learning experiences from the essentials and from the class. Do not forget to document limitations and implications for future research/practice. Please review the PowerPoint prior to submitting your assignment, thank you.
Amino Acids and Proteins
Structure of -amino acids
The 20 Amino Acids Found in Proteins
Formation of a Peptide
Polypeptide backbone
9.bin
10.bin
Proteins are made of 20 amino acids linked by peptide bonds
Polypeptide backbone is the repeating sequence of the N-C-C-N-C-C… in the peptide bond
The side chain or R group is not part of the backbone or the peptide bond
ProteinsMake up about 15% of the cellHave many functions in the cellEnzymesStructuralTransportMotorStorageSignalingReceptorsGene regulationSpecial functions
Motor- myosin
Storage- ferritin, transport- hemoglobyn
*
Importance of ProteinsMain catalysts in biochemistry: enzymes (involved in virtually every biochemical reaction)Structural components of cells (both inside and outside of cells in tissues)Regulatory functions (if/when a cell divides, which genes are expressed, etc.)Carrier and transport functions (ions, small molecules)
Levels of Protein StructurePrimary Structure - amino acid sequence in a polypeptide
Secondary Structure - local spatial arrangement of a polypeptide’s backbone atoms (without regard to
side chain conformation)
Tertiary Structure - three-dimensional structure of entire polypeptide
Quaternary Structure - spatial arrangement of subunits of proteins composed of multiple polypeptides (protein complexes)
3-D Structure of Myoglobin
People with proteinuria have urine containing an abnormal amount of protein. The condition is often a sign of kidney disease.
Healthy kidneys do not allow a significant amount of protein to pass through their filters. Kidney disease often has no early symptoms. One of its first signs may be proteinuria that's discovered by a urine test done during a routine physical exam. Blood tests will then be done to see how well the kidneys are working.
Both diabetes and high blood pressure can cause damage to the kidneys, which leads to proteinuria.
Proteinuria (Protein in Urine)
Proteinuria (Protein in Urine)
Methods of Protein Estimation
Quantitative
Biruet methodBradford methodFolin-Lowry methodKjeldahl methodBicinchoninic acid method (BCA method)UV methodFlourimetric methodMass spectrometry
Protein Determination assay
Bicinch ...
A new precise accurate and reliable validated method for the determination of Capecitabine was developed by using
reverse phase high performance liquid chromatography in pharmaceutical dosage forms. Spectrophotometer
determination was carried out at an absorption maximum of 240nm by using methanol. The linearity was over the
concentration range of 20-120 μg/ml with correlation coefficient 0.999. Chromatographic separation was carried
out by using a mobile phase of methanol: Acetonitrile: water (80:20:80 V/V) on Waters 2487 dual absorbance
column in an isocratic mode at a flow rate of 1.1 ml/min with UV detection at 240 nm. The developed methods were
found to be precise and accurate for the estimation of Capecitabine in pharmaceutical dosage forms and could be
used for routine analysis.
Keywords: Capecitabine, RP-HPLC, Spectrophotometry, Waters 2487 dual absorbance detector, Nova pack 300 ×
3.9mm 5μ as column, 240nm
Pharmaceutical Biotechnology Research Presentation : Recombinant Streptokinase
Dr. Godfrey Mazhandu
Professor Peivand Pirouzi Inc. -
Copyright 2015 - Professor Peivand Pirouzi Inc., International Corporate Training, Canada
All rights reserved
Determination of Total Protein Using the LAMBDA UV/Vis SpectrophotometerPerkinElmer, Inc.
The Lowry and Biuret methods are standard methods for protein quantification. Though the latter is more sensitive and is used for investigative work, it is limited by (1) poor stability of the combined reagent, (2) non-reproducibility of color, especially at low protein concentrations, and (3) a non-linear chromogenic response with protein concentration. Ohnishi and Barr1 modified and simplified the Biuret combined reagent for the Lowry procedure and at the same time improved its stability. This application note describes the modified Lowry procedure for protein analysis.
Principles and methods of different microbiological assay, methods for standa...Ms. Pooja Bhandare
PHARMACEUTICAL MICROBIOLOGY (BP303T)Unit-IV Part-2 Principles and methods of different microbiological assay, methods for standardization of antibiotics.
Introduction: Principles Advantages of Microbial Assay: Disadvantages of Microbial Assay: MICROBIOLOGICAL ASSAY OF ANIBIOTICS PRINCIPLE Media used for antibiotics assay Standard Preparation. Buffer Solutions Preparation of the Sample Solution: Test Organisms Preparation of inoculum: Methods of preparation of test organism suspension: Assay Methods: Method A: Cup-plate or Cylinder Plate Method.
Method B: Turbidimetric or Tube assay Method
Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-...SriramNagarajan15
A new, simple sensitive, rapid, accurate and precise RP-HPLC method was developed for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Clopidogrel bisulphate was chromatographed on a reverse phase C18column (150 mm x 4.5 mm, i.d 5μm) in a mobile phase consisting of acetonitrile and phosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow rate of 1 ml/min with detection at 224 nm. The detector response was linear in the concentration of 50-150 μg /ml. The limit of detection and limit of quantitation was found to be 1.3 and 4.2 µg/ml, respectively. The intra and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was 99.79%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Key words: Clopidogrel bisulphate, RP-HPLC, Validation, Accuracy, Precision.
Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-...SriramNagarajan15
A new, simple sensitive, rapid, accurate and precise RP-HPLC method was developed for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Clopidogrel bisulphate was chromatographed on a reverse phase C18column (150 mm x 4.5 mm, i.d 5μm) in a mobile phase consisting of acetonitrile and phosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow rate of 1 ml/min with detection at 224 nm. The detector response was linear in the concentration of 50-150 μg /ml. The limit of detection and limit of quantitation was found to be 1.3 and 4.2 µg/ml, respectively. The intra and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was 99.79%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Key words: Clopidogrel bisulphate, RP-HPLC, Validation, Accuracy, Precision.
Similar to Practical 3 Quantitative determination of protein concentration using spectrophotometry (20)
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Practical 3 Quantitative determination of protein concentration using spectrophotometry
1. FACULTY OF INFORMATION SCIENCE AND
TECHNOLOGY
Practical 3
Quantitative determination of protein concentration using
spectrophotometry
HBC 1011
Biochemistry I
Trimester 1 2018/2019
Name: MOHAMED REDA
ID: 1151303266
2. Introduction
Accurate protein quantitation is essential to all experiments related to proteins in a
multitude of research topics. Different methods have been developed to quantitate proteins in a
given assay for total protein content and for a single protein. Total protein quantitation methods
comprise traditional methods such as the measurement of UV absorbance at 280 nm,
Bicinchoninic acid (BCA) and Bradford assays, as well as alternative methods like Lowry assay.
In this practical, the method of Bradford is used to determine the protein concentration. The
Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration
of protein in a solution. It is based on the amino acid composition of the measured protein. The
protein will form a complex with the coomassie blue dye. The protein concentration can be
evaluated by determining the amount of dye in the blue ionic form and by measuring the
absorbance of the solution at 595 nm using a spectrophotometer. While using the Bradford assay,
detergent containing buffer must be avoided as it will disrupt the coomasie dye and produce an
inaccurate result.
3. Methodology
Part A: Construction of linear graph from known protein standards
1) Seven Eppendorf tubes were prepared and labeled as blank and from 1 to 6.
2) An amount of 1ml of dye reagent and 20µl of PBS was pipetted into an Eppendorf tube
labeled blank and was mix by inverting few times.
3) Appropriately labeled Eppendorf tubes were added with 20µl of pre-diluted standard and 1ml
of dye reagent by using a micropipette.
4) The spectrophotometer was set to 595nm and the instrument was set to zero on a 1.5ml
polystyrene cuvette fill with solution from Eppendorf tube labeled blank.
5) The solution was poured back to the Eppendorf tube and the next set of solution was added to
the empty cuvette.
6) The absorbance of all the standard were measured.
7) A linear standard curve was constructed by plotting A595 values of the known standards
against the concentration of standard protein.
Part B: Quantitative determination of protein in food samples
1) A food sample is prepared for dilution.
2) The food sample was diluted with dilution factor of 10x, 50x and 100x and was pipetted in to
the Eppendorf tubes labeled with corresponding dilutions.
3) An amount of 1ml of dye reagent and 20µl of PBS was pipetted into an Eppendorf tube
labeled blank and was mix by inverting few times.
4) An amount of 20µl of each diluted samples and 1ml of dye reagent were pipetted into the
new Eppendorf tubes labeled with corresponding dilutions.
5) The spectrophotometer was set to 595nm and the instrument was set to zero on a 1.5ml
polystyrene cuvette fill with solution from Eppendorf tube labeled blank.
6) The solution was poured back to the Eppendorf tube and the next set of solution was added to
the empty cuvette.
7) The absorbance of each diluted food samples were recorded.
4. 8) Food sample with dilution factor 50x was chosen to be replicated to obtain the average
protein concentration for a more accurate result.
9) Final concentration of the food sample is calculated by multiplying the protein concentration
obtained from the graph with the dilution factor to get the actual concentration.
10) The final concentration of food sample and the actual protein content information was
tabulated and the standard deviation was calculated.
5. Result and Discussion
Table 1: Standard Curve Absorbance Values
Standard Diluent volume
(PBS -µl)
Source of
Standard
(mg/ml)
Standard
volume (µl)
Final
volume (µl)
Final
concentration of
BGG (mg/ml)
1 0 2 100 100 2.000
2 50 2 150 200 1.500
3 10 2 100 200 1.000
4 100 1.5 100 200 0.750
5 100 1 100 200 0.500
6 100 0.5 100 200 0.250
By using the formula of M1V1 = M2V2 the amount of standard volume to be used for each
standard are calculated. The remaining volume was then top up with the diluent which is the PBS
solution to obtain the exact final volume and the final concentration.
Table 2: Protein Standard Absorbance
Standard Final concentration of BGG
(mg/ml)
A595 Average A595
A B C
1 2.000 1.344 1.340 1.143 1.276
2 1.500 1.012 0.937 0.944 0.964
3 1.000 0.711 0.702 0.680 0.698
4 0.750 0.558 0.624 0.531 0.571
5 0.500 0.459 0.424 0.358 0.414
6 0.250 0.175 0.177 0.191 0.181
The reason for obtaining duplicate result of the same standard is to ensure that the result
will be accurate since the pipetting skill of each individual was different. Some of them might
6. have done error during the pipetting process. There are several ways to reduce the error in
pipetting. For example, if you need to dispense 15 µl of solution, a 1ml pipette would be the
wrong choice, whereas a 20 µl pipette would be ideal.
Table 3: Dilution of protein samples
(Soybean Sample)
Dilution Protein Sample (µl) Diluent Volume (PBS
- µl)
Final volume (µl)
10x 20µl of original
protein sample
180 200
50x 40µl of 10x protein
sample
160 200
100x 100µl of 50x protein
sample
100 200
For diluting the protein samples given, dilution factor method was applied to obtain the
desired concentration. Firstly, final volume to be obtain was set. To dilute the protein sample into
10x, 20µl of the original sample was used and the remaining volume was top up with the diluent,
PBS solution. Then obtained 40µl of solution from the 10x diluted sample and the remaining
volume was top up with diluent to create a 50x diluted sample. Lastly, 100µl of 50x diluted
protein sample was obtained and mixed with 100µl of PBS solution to get a final volume of
200µl and 100x dilution.
7. Table 4: Spectrophotometric Data for Protein Samples
Sample A595 Average
A595
Dilution
Factor
Protein
Concentration
(ml/ml)
Average Protein
Concentration
(mg/ml)
X1 0.440
0.435 50x
0.541
0.533X2 0.447 0.553
X3 0.417 0.505
Y1 0.625
0.660 10x
0.856
0.925Y2 0.684 0.972
Y3 0.671 0.947
Z1 0.337
0.331 50x
0.417
0.411Z2 0.324 0.403
Z3 0.332 0.412
The protein concentration of food samples were obtained by referring to the standard
linear graph with the known value of absorbance which was obtained by measuring the
absorbance of protein samples. The average of absorbance and protein concentration was
calculated to get a more precise reading.
Table 5: Comparing measured protein concentrations to the values found on food labels
Protein Samples Bradford Assay (mg/ml) Final
Concentration from Table 4
(mg/ml)
Food Label (mg/ml)
X
(Soybean Milk)
26.65 22
Y
(Prebiotic Fermented Milk)
9.25 11
Z
(Full Cream Milk)
20.55 34
8. Table 6: Standard deviation of average protein concentration from different sample
Protein Samples Average Protein
Concentration (mg/ml)
Standard Deviation of Average
Protein Concentration
X
(Soybean Milk)
0.533
0.269Y
(Prebiotic Fermented Milk)
0.925
Z
(Full Cream Milk)
0.411
Graph 1: The relationship between Concentration of Standard and A595
Based on the standard linear graph, the experimentally actual protein concentration of
soybean milk is 26.65 mg/ml while for prebiotic fermented milk is 9.25mg/ml and lastly full
cream milk having 20.55 mg/ml. Compared to the actual values on the food label, the result
obtained experimentally is more than the actual value. This can be due to errors in preparing the
diluted protein samples. Possibly more protein samples volume are pipetted without notice
during the dilution process. Therefore acquiring an inaccurate result. For prebiotic fermented
milk, the experimental value is quite close to the actual result. For full cream milk, the
9. experimentally obtained protein concentration was far lesser than the actual value. This might be
due to errors in preparing the diluted protein samples where the dilution factor method applied
might be some mistake in it. From the food label we know that the highest protein concentration
food should be full cream milk while the lowest is the prebiotic fermented milk and the
intermediate is soybean milk. Yet the experimental value obtained was quite different whereby
the highest is the soybean milk and the intermediate is the full cream milk. This could be the
result of lacking pipetting skill and errors in calculating for diluting protein samples.
Conclusion
In conclusion, protein concentration of food samples can be determine by using different
type of method correspond to their requirement.
10. Question
1) What are the functions of spectrophotometer?
Spectrophotometer is used to measure the concentration of the solution by determining
the absorbance, identifying the organic compounds by determining the maximum
absorption and used for color determination within the spectral range.
2) Why is it important to know the linear range of protein standard in protein quantitate on
assay? What do you need to do if the absorbance of the protein is out of the linear range?
it is important to know the linear range of protein standard in protein quantitate on assay
because if you have the standard curve you will be able to identify the concentration of
any unknown protein sample. if the absorbance of the protein is out of the linear range
then you should see If the standard curve is leveling off, then you should not use the
points with the higher absorbance.
3) The protein concentration you obtained from the experiment may be different from the
value on the food label. Explain this observation.
This may be due to error in pipetting th perotein solution when performing the dilution
procedure and transferring the solution. Difference of the protein concentration may also
be due to the uncalibrated micropipette.
4) How long can the samples sit before being read? Is it alright if you measure the sample
absorbance after 1 hour? Why?
The readings will be inaccurate if the sample is let to sit for too long. This is because the
protein molecules in the protein sample will eventually sediment on the base of the
container. Thus the absorbance reading will be inaccurate.
5) You are using bovine gamma globulin as the protein standard, but you are measuring
protein concentration of samples from other sources. What is the assumption you make
for determination of the protein concentration of samples from other sources?
We assume that the standard protein has no errors and it produce a perfect result, the best
11. fit linear graph, and we use it as a reference to the protein that are going to be tested to
find the protein concentration based on the known absorbance value.
6) How do you know the protein absorbance is out of the linear range? Should you use these
values when plotting the graph? Why?
The linear range of the protein absorbance is between 0.1 and 1.0. The Other values
lower or higher than that will be consider out of range. We can use the values, to obtain
sufficient points, to plot the graph but when referring to the graph to obtain the protein
concentration with known absorbance, the value in between the linear range is only to be
used. Any value exceeding the range can be inaccurate.
7) You purified a protein which is eluted in a buffer containing detergent. Which protein
concentration determination method will you use? Why?
BCA assay. It is because this method is not sensitive to detergent. An accurate result can
be obtained. Bradford assay is not used because Bradford assay is sensitive to the
detergent it interferes with the coomasie dye.
8) You purified a protein which is eluted in a buffer containing reducing agent. Which
protein concentration determination method will you use? Why?
Bradford assay. It is because Bradford assay is not affected by the presence of reducing
agents. Lowry assay and BCA assay are sensitive to reducing agent which interfere the
protein thus it will affect the result.