Bradford Assay_lab

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…

(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.

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.

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.

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.

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

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.

List 4 advantages of biological value of protein determination method.

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.

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.

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…

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

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.

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

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.)

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.

Draw the schematic diagram of the protein purification through hydrophobic column chromatography and explain the purpose of each step.

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.

SDS-PAGE gels are useful in determining the molecular weights of proteins; however, the molecular weights of are usually not reliable. protei

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)?

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

Compare and contrast the following protein characterization techniques in terms of the principles governing their functions.    size exclusion chromatography vs ion-exchange chromatography

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…

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?

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.

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.

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?

Compare and contrast the following protein characterization techniques in terms of the principles governing their functions.    isoelectric precipitation vs salting out process

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…

Explain Peptide Mapping and Internal Sequencing of Proteins from Acrylamide Gels.

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.

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.