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Bradford Assay Lab Bio3700 Introduction: A protein assay is a laboratory technique used to determine the concentration of a specific
protein in a sample. There are four main types of protein assays in regard to quantification: Bradford, Lowry, BCA, and UV. Many purchased products contain labels displaying the amount
of protein in the product. In this laboratory experiment, the accuracy of a muscle milk protein description is tested using the Bradford protein assay technique. Using Coomassie Brilliant blue dye G-250, the binding of the dye to basic and aromatic amino acids with a particular affinity to arginine can be seen as the color shifts from a red to a bluer version. There is an absorbance shift
under acidic conditions. The results obtained from the Bradford assay can vary slightly from one
protein to the next. This technique is very good at avoiding most interference from other reagents. Addition of SDS to the sample should be avoided, because detergents, as such, denatures the proteins. Using bovine serum albumin (BSA), a standard graph can be formed, and from the calibration curve, the amount of protein in the muscle milk can be determined. This calculation will show the accuracy of the company’s protein label. Results: Below in Table 1, the raw and corrected absorbances of the BSA Standard are arranged in
a table. These values were used to create the BSA Standard Curve graph. The raw and corrected absorbances of the muscle milk and the corresponding dilutions can be found in Table 2 below. Using the slope of the BSA Standard Curve seen in Figure 1 below, the concentration of the 1:500 protein dilution was 0.165 mg/ml. This value was used to find the amount of protein in the muscle milk sample in the calculations shown below. y=1.3585x
x= 0.165 mg/ml 0.165mg/ml X 500 mg/ml = 82.5 mg/ml This value is then multiplied by the amount of muscle milk in the bought container (ml). 82.5 mg/ml X 330 ml = 27225 mg/ml = 27.2 g/ml protein The calculations show that there was a measurement of 27.2 g/ml of protein found in the tested muscle milk sample.
Discussion: Based on the muscle milk creator’s printed label, the muscle milk should contain 25 g/ml of protein in a 330 ml container. The results obtained from this lab state that there is roughly 27.2
g/ml of protein in the container. Based on the R
2
value, 0.9212, from the BSA Standard Curve graph (Figure 1), it can be concluded that the experiment most likely contained little error. The experimental value of protein is not far off from the company’s printed value, and with the R
2
value being high, the assumption that the product contained at least 25 g/ml of protein can be claimed accurate. Supplemental: BSA Standard (mg/ml) Raw Absorbance Corrected Absorbance 0
0.307
0
0
0.315
0
0
0.320
0
0.05
0.403
0.096
0.05
0.365
0.058
0.05
0.386
0.079
0.1
0.312
0.005
0.1
0.312
0.005
0.1
0.307
0
0.2
0.583
0.276
0.2
0.632
0.325
0.2
0.602
0.295
0.3
0.820
0.513
0.3
0.806
0.499
0.3
0.808
0.501
0.4
0.856
0.549
0.4
0.885
0.578
0.4
0.866
0.559
0.5
0.862
0.555
0.5
0.977
0.670
0.5
0.945
0.638
Table 1: BSA standard versus Absorbance Values Protein Dilution Raw Absorbance Corrected Absorbance Undiluted 2.586
2.279
Undiluted 2.521
2.214
Undiluted 2.561
2.254
1:10
1.895
1.588
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Related Questions
Simple problem-solving: Imagine you did a Bradford assay, and measured the absorbance of a set of protein standards and two unknowns. You repeated each measurement three times, and got the following readings in the table below.
Concentration (micrograms/ml)
A595 -
trial 1
A595- trial 2
A595- trial 2
average A595
A595 of sample minus A595 of blank
0
1.501
1.446
1.447
1.465
0
2
1.624
1.558
1.559
1.580
0.115
5
1.731
1.749
1.712
1.731
0.266
10
1.901
1.838
1.892
1.877
0.412
15
2.161
2.108
2.228
2.166
0.701
18
2.231
2.277
2.319
2.276
0.811
unknown 1 - D1
1.717
1.713
1.644
1.691
0.226
unknown 2 - D1
1.668
1.649
1.656
1.658
0.193
concentration of unknown 1
concentration of unknown 2
Calculate the concentration of the unknowns and answer the following question:
Did you need to do any manipulations before applying the…
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(b) Both laboratories used 10 micrograms of protein each in their kinetic assays. Protein concentrations weredetermined by the Bradford protein assay. Assay conditions employed in the two labs (pH, temperature,etc.) were also identical. What would be the most plausible cause for the discrepancy in the Vmax valuesfor the compound I? Explain.Recall that the Bradford assay measures total protein amounts in sample solution based on complexformation between a dye and proteins. Also, the assay solution used in both labs does not contain anyinhibitors.
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pls explain
Increasing the saturation of the ammonium sulfate is a prerequisite in isolating a target protein that is rich in Cys and Tyr residues. Which of the following techniques should be considered in accurately quantifying the isolated protein?I. Running the isolated protein in a dialysis or GFC set up.II. Using Biuret or BCA assay as the colorimetric quantitation method.III. Using Bradford or Lowry assay as the colorimetric quantitation method.A. I onlyB. II onlyC. I and IIID. I, II and III. Bradford Assay is most suitable to use when the extraction buffer is below the target protein’s pI. This is so because the protein would be morea. Positively charged allowing the CBB G-250 dye to bind via its sulfonate groups.b. Negatively charged allowing the CBB G-250 dye to bind via its sulfonate groups.c. Neutrally charged allowing the CBB G-250 dye to bind via its sulfonate groups.d. Zwitterionic allowing the CBB G-250 dye to bind via its sulfonate groups.
arrow_forward
What are the advantages of gel filtration as a technique for protein purification?
Identify two types of gels (sieving matrix) that may be used for gel filtration chromatography and discuss the types of samples which may be used for each.
Discuss the principle behind affinity chromatography and differentiate it from gel filtration chromatography.
arrow_forward
A good way to increase total proteome coverage without using 2D PAGE is to:
"Use two, orthogonal types of chromatography"
Enrich for phosphopeptides only
Analyze whole proteins instead of proteolytic peptides.
arrow_forward
If the target protein is 0.1% of the total protein in the original mixture, a three-step purification process requires:
Group of answer choices
a 100-fold purification at each step
a 22-fold purification at each step
a 10-fold purification at each step
a 5-fold purification at each step
arrow_forward
Mass spectrometry is a powerful tool in proteomics. What are the four key features of a mass spectrometer? Describe briefly how MALDI and two-dimensional polyacrylamide gel electrophoresis could be used to identify a protein expressed in cancer cells but not in normal healthy cells.
arrow_forward
List 4 advantages of biological value of protein determination method.
arrow_forward
Please compare the design and operation of a protein precipitation unit to that of a chromatography operation for the objective of protein purification at industrial scale. Please supply at least three qualitative factors and three quantitative measures for your comparison. Please make sure your comparison focuses on the effectiveness and efficiency of the downstream processing unit operations.
arrow_forward
Using the SDS-PAGE and the calibration curve given, calculate the size of the lysozyme and ovalbumin bands from this lysozyme purification. Lysozyme is the bottom band in the samples, ovalbumin is the most intense band in the first three samples, and the first and last lanes are molecular mass standards.
arrow_forward
Experiment: Bradford protein assaygive the answers (4-5 lines) of review questions in the end. the answer should be logical and understandable and without plagiarism. avoid copy-pasting. PLEASE GIVE THE ANSWER OF 3rd AND 4th QUESTION. ITS COMPULSORY.Background information:The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis. The method described below is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg protein. Scale down the volume for the "micro assay procedure," which uses 1 ml cuvettes. Protocols, including…
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Exercise 1
Summarize the data below obtained from the protein experiments for determination of protein
concentration in the form of a graph. Plot a graph containing a title and labels for both of the
axis. Three readings were taken for each protein concentration. Find the averages and standard
deviations for each reading and plot your graph using Microsoft EXCEL, complete with the error
bars. This graph represents
will be used to measure the protein concentration in an unknown protein solution.
calibration curve for a protein assay (next experiment) where this
Concentration of protein
(Hg/mL)
Absorbance, 595 nm
1
2
3
0.04
0.05
0.03
2
0.12
0.11
0.14
5
0.26
0.25
0.25
10
0.49
0.49
0.51
25
0.82
0.85
0.83
50
1.28
1.24
1.25
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A protein gives a single band on SDS gel electrophoresis, as shown in lanes 1 and 2 below. There is little, if any, effect from adding β-mercaptoethanol(BME) to the sample; if anything, the protein runs a little bit slower. When treated with the proteolytic enzyme thrombin and electrophoresis in the absence of BME, the protein migrates a bit more rapidly (lane 3). But if BME is present, two much more rapidly migrating bands are found (lane 4). Explain these results in terms of a model for the protein.
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You have an aqueous solution containing: Alaine (a mono-amino, monocarboxylic acid) Fructose (a non-ionic monosaccharide) Glycogen (a non-ionic large polysaccharide) Ribose-5-phosphate (an anionic monosaccharide phosphate) t-RNA (a polyanionic nucleic acid, MW~ 30,000). Assuming you have a distinctive assay for each of these compounds, what procedures would you use to obtain gram quantities of each of these compounds free of each of the other compounds
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In Multi-Column Purification of rGFP.
What happens to the protein amount, protein purity, and/or specific activity of a purification fraction if one of the three is changed? (i.e. understand the relationship between the three.)
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Subject: Biochemistry, chemistry, polyacrylamide gel electrophoresis
Differentiate native PAGE and SDS-PAGE in terms of the relative molecular weight of protein bands obtained. Explain your answer.
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Draw the schematic diagram of the protein purification through hydrophobic column chromatography and explain the purpose of each step.
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Under most in vitro assay conditions, the enzyme is used in catalytic amounts
(10 to 10" M). Estimate the concentration of an enzyme in a living
cell. Assume that (a) fresh tissue is 80% water and all of it is intracellular, (b)
the total soluble protein in a cell represents 15% of the wet weight, (c) all the
soluble proteins are enzymes, (d) the average molecular weight of a protein is
150,000, and (e) about 1000 different enzymes are present.
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SDS-PAGE gels are useful in determining the molecular weights of
proteins; however, the molecular weights of
are usually not reliable.
protei
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what are the advantages and disadvantages of using recombinant protein and affinity chromatography for protein purification compared to gel filtration (size exclusion chromatography) and DEAE-sepharose chromatography (ion-exchange chromatography)?
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Compare and contrast the following protein characterization techniques in terms of the principles governing their functions.
size exclusion chromatography vs ion-exchange chromatography
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You start with 100 units of protein activity and 100 grams of total protein.
After the first centrifugation step, you have 25% less activity and 85% less
total protein. What is your fold-purification of the target protein?
0.3-fold
3.4-fold
75-fold
5-fold
20-fold
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Pls explain If the voltage were NOT increased at the start of the resolving gel run, the overall experiment time will ______, while the calculated electrophoretic mobilities will ______.a. increase, increaseb. increase, decreasec. decrease, increased. decrease, decreasePick all that are TRUE regarding analysis of quaternary structures of proteins using polyacrylamide electrophoresis:I. The added β-mercaptoethanol disrupts S--S bonds bridging the polypeptide chains causing the appearance of higher Rf bands compared to the native protein run. II. Heating up any protein before subjecting to SDS-PAGE will always result in the formation of more than one band.III. A good asymmetrical gel layout would be : (Lane 1) MW ladder, (2) native protein, (3) protein + β-ME, (4) protein + HCL, (5) protein + β-ME + HCl.IV. Formation of a single band in the protein + β-ME + HCl run, whose Rf is lower than the native run, could be indicative that the protein is a homodimer.A. I onlyB. I and IIC. II and…
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Saturation experiments using radioligand is one method of determining the affinity of a ligand to a drug target.
What are other methods used in high-throughput screening (HTS) of compound libraries that do not use a radioligand?
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Design an assay with purified phycocyanin to test protein stability using 500ml of 95% ethanol. Then do the same with 2 M sodium chloride. Make each step clear.
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A protein gives a single band on SDS gel electrophoresis, as shown inlanes 1 and 2 below. There is little, if any, effect from adding β-mercaptoethanol (BME) to the sample; if anything, the protein runs a little bitslower. When treated with the proteolytic enzyme thrombin and electrophoresis in the absence of BME, the protein migrates a bitmore rapidly (lane 3). But if BME is present, two much more rapidlymigrating bands are found (lane 4). Explain these results in terms of amodel for the protein.
arrow_forward
Affinity chromatography
You have created a fusion protein tagged with Glutathione-S-Transferase (GST). Your lab mate tells you that the affinity columns for this type of tagged protein are very similar to that of Histadine tagged proteins.
Using the following elements set up a purification column and construct a protocol for an affinity purification using this tag.
A large amount of glutathione is usually used to elute the tagged protein off the column. How might this work?
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We discussed various assay attributes in class which provide insight into the effectiveness or suitability of a given analytical method. As part of assay development, various attributes were investigated. For each description below, indicate which assay attribute was being evaluated (partial list of attributes include precision, accuracy, specificity, linearity, LOQ/LOD, etc.).
The impact of assay temperature and pH on assay results were evaluated to find a suitable range of conditions for the activity assay.
Various known concentrations of standard α-AM solutions were evaluated, and the proportionality of the responses were determined.
A reference standard of known α-AM (alpha-amylase) concentration was evaluated in triplicate to determine the variability (or scatter) of repeat measurements.
Spiked standard of known specific activity and purity was spiked into a sample and the ability for the assay to measure the true biological activity was determined.
The impact of various extract…
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Compare and contrast the following protein characterization techniques in terms of the principles governing their functions.
isoelectric precipitation vs salting out process
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Explain Peptide Mapping and Internal Sequencing of Proteins from Acrylamide Gels.
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Example. A new plant species was discovered. To determine the protein content of the plant, an adequate amount of the plant extract was acquired, and bradford assay was performed. The absorbance reading of the plant extract is 0.272. What is the protein concentration, in μg/L, of the plant extract? A bovine serum albumin (BSA) Stock solution with a concentration of 250 μg/mL was used and mixtures with the following compositions and absorbance reading were prepared in picture.
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Which technique will be most suitable for desalting the protein or analyzing the actual dimensional (D) native form and what would be the advantages of this selection? Discuss two to three supportive evidence of the technique application.
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Related Questions
- Simple problem-solving: Imagine you did a Bradford assay, and measured the absorbance of a set of protein standards and two unknowns. You repeated each measurement three times, and got the following readings in the table below. Concentration (micrograms/ml) A595 - trial 1 A595- trial 2 A595- trial 2 average A595 A595 of sample minus A595 of blank 0 1.501 1.446 1.447 1.465 0 2 1.624 1.558 1.559 1.580 0.115 5 1.731 1.749 1.712 1.731 0.266 10 1.901 1.838 1.892 1.877 0.412 15 2.161 2.108 2.228 2.166 0.701 18 2.231 2.277 2.319 2.276 0.811 unknown 1 - D1 1.717 1.713 1.644 1.691 0.226 unknown 2 - D1 1.668 1.649 1.656 1.658 0.193 concentration of unknown 1 concentration of unknown 2 Calculate the concentration of the unknowns and answer the following question: Did you need to do any manipulations before applying the…arrow_forward(b) Both laboratories used 10 micrograms of protein each in their kinetic assays. Protein concentrations weredetermined by the Bradford protein assay. Assay conditions employed in the two labs (pH, temperature,etc.) were also identical. What would be the most plausible cause for the discrepancy in the Vmax valuesfor the compound I? Explain.Recall that the Bradford assay measures total protein amounts in sample solution based on complexformation between a dye and proteins. Also, the assay solution used in both labs does not contain anyinhibitors.arrow_forwardpls explain Increasing the saturation of the ammonium sulfate is a prerequisite in isolating a target protein that is rich in Cys and Tyr residues. Which of the following techniques should be considered in accurately quantifying the isolated protein?I. Running the isolated protein in a dialysis or GFC set up.II. Using Biuret or BCA assay as the colorimetric quantitation method.III. Using Bradford or Lowry assay as the colorimetric quantitation method.A. I onlyB. II onlyC. I and IIID. I, II and III. Bradford Assay is most suitable to use when the extraction buffer is below the target protein’s pI. This is so because the protein would be morea. Positively charged allowing the CBB G-250 dye to bind via its sulfonate groups.b. Negatively charged allowing the CBB G-250 dye to bind via its sulfonate groups.c. Neutrally charged allowing the CBB G-250 dye to bind via its sulfonate groups.d. Zwitterionic allowing the CBB G-250 dye to bind via its sulfonate groups.arrow_forward
- What are the advantages of gel filtration as a technique for protein purification? Identify two types of gels (sieving matrix) that may be used for gel filtration chromatography and discuss the types of samples which may be used for each. Discuss the principle behind affinity chromatography and differentiate it from gel filtration chromatography.arrow_forwardA good way to increase total proteome coverage without using 2D PAGE is to: "Use two, orthogonal types of chromatography" Enrich for phosphopeptides only Analyze whole proteins instead of proteolytic peptides.arrow_forwardIf the target protein is 0.1% of the total protein in the original mixture, a three-step purification process requires: Group of answer choices a 100-fold purification at each step a 22-fold purification at each step a 10-fold purification at each step a 5-fold purification at each steparrow_forward
- Mass spectrometry is a powerful tool in proteomics. What are the four key features of a mass spectrometer? Describe briefly how MALDI and two-dimensional polyacrylamide gel electrophoresis could be used to identify a protein expressed in cancer cells but not in normal healthy cells.arrow_forwardList 4 advantages of biological value of protein determination method.arrow_forwardPlease compare the design and operation of a protein precipitation unit to that of a chromatography operation for the objective of protein purification at industrial scale. Please supply at least three qualitative factors and three quantitative measures for your comparison. Please make sure your comparison focuses on the effectiveness and efficiency of the downstream processing unit operations.arrow_forward
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