PAGE and Western Blot Practice Sheet_Answer Key

Activity 1 Activity 2 Activity 3 Activity 4

When the gel is removed from the chamber in SDS-PAGE, only the “pre-stained” protein standard is visible. There are no other proteins visible on the rest of the gel. Did something go wrong here? Why can’t you see the other proteins?

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Electrophoresis     A protein required 6.8 min to travel 82 cm to the detector in a 96 cm -long capillary tube with 25.4 kV between the ends. Find the apparent electrophoretic mobility.

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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

(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.

Part B/D?

A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the native protein has a molecular weight of 300,000. Chromatography of the protein in the presence of 6 M guanidine hydrochloride (a denaturant) yields a single peak corresponding to a molecular weight of 50,000. Chromatography of the protein in the presence of 6 M guanidine hydrochloride and 10 mM beta-mercaptoethanol (a disulfide bond reductant) yields peaks corresponding to molecular weights of 30,000 and 20,000. What does this data tell you about the structure of this protein? (Be thorough in your answer!)

What is the function of sodium dodecyl sulfate (SDS) in SDS-PAGE? stabilizes the gel matrix, improving resolution during electrophoresis SDS solubilizes proteins to give them uniformly negative charges, so the separation is based purely on size. SDS raises the pH of the gel, separating multiunit proteins into individual subunits. SDS solubilizes proteins to give them uniformly positive charges, so separation is based purely on pH.

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.

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.

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.

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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Marker Proteins Molecular Weight (daltons) Distance 132,000 66,000 43,000 14,400 Serum Albumin (Dimer) Serum Albumin (Monomer) 23mm 32mm Ovalbumin 39mm 60mm Lysozyme Unknown 1 26mm Unknown 2 58mm Rabbit serum (albumin) 33mm 1.On semilog paper (provided below) plot the distance migrated by each standard protein as a function of molecular weight. Determine the molecular weight of the unknown proteins and the major protein found in rabbit serum (albumin) from this calibration curve. ii) Interferon is a protein of 25,000 Dalton. How far would it have migrated, land it been included in this experiment? iii) The average amino acid residue in a protein has a molecular weight of 120 Daltons. Assuming that your unknowns are average, how many amino acid residues do they contain

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PLEASE have the following and help D:. What i need for the chosen of 2/3 choices.  2)   Illustrate detailed chemical scheme for how you will functionalize your biomolecule to the surface of the sensor (e.g. if you wanted to functionalize a protein to a gold SPR chip, you should could show the assembly of an amine-terminated SAM, followed by carbodiimide coupling of. the protein). These schemes should be drawn using a chemical illustration software package (ChemDraw / ChemSketch).Your chemical schemes should be as detailed as necessary to accurately depict the chemical design of your system, DO NOT INCLUDE a step-wise mechanism, it is not necessary. You should include at least one literature reference to support your conjugation strategy.3)  Include an illustration of the functional sensor, and describe whether the sensor is label-free or not, what signal is observed, and how you would detect the interaction (i.e. if a labeled antibody is required, you should describe what label would…

A mixture containing aspartic, lysine, serine and tyrosine was subjected to gel electrophoresis at pH 7.0. Indicate the position of each amino acid in the gel. (Show comprehensive explanation, and with graphical illustration if possible).

Don't give AI generated solution  The genomic DNA information on bovine FABP4 gene is listed below: source 1..4389 /organism="Bos taurus" /mol_type="genomic DNA" /chromosome="14" gene 1..4389 /gene="FABP4" /note="fatty acid binding protein 4, adipocyte mRNA join(1..133,2843..3015,3608..3709,4173..4389) /gene="FABP4" /product="fatty acid binding protein 4, adipocyte" CDS join(61..133,2843..3015,3608..3709,4173..4223) /gene="FABP4"    Give correct typing answer with explanation

A protein of unknown structure has been purified. Size-exclusion chromatography reveals that the native protein has a MW of 240,000. Chromatography in the presence of 6 M guanidine hydrochloride yields a single peak corresponding to a protein of MW 60,000. Chromatography in the presence of 6 M guanidine hydrochloride and 10 mM B-mercaptoethanol yields peaks for proteins of MW 34,000 and 26,000. Explain what can be determined about the structure of this protein from these data.

Peptide Sequencing Answer numbers 1 and 2. For number 2, show the OVERLAP method.

272-2 - 6. The protein assay kit that we will use to quantitate horseradish peroxidase requires absorbance measurements at 562 nm. What type of radiation is this? CCT CCT CCT GOT CCT CCT C Read through the document describing the BCA Protein Assay.? This document is posted separately on Blackboard. What is the protein used in the assay? Why is the assay protein used instead of horseradish peroxidase? 7.

Western blotting is a routine technique in protein analysis that provides qualitative and semi- quantitative data. In this work, protein separation is first carried out using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). (i) Explain the principle of SDS-PAGE in protein separation. After the electrophoretic separation, the proteins are transferred to a nitrocellulose membrane prior to staining with antibodies specific for the protein understudy. Describe in detail how the primary and secondary antibodies could aid in the protein detection process. (ii)

The OXA-M290 protein is next purified by size exclusion chromatography. To determine the best type of size exclusion resin to use, the size of OXA-M290 must first be determined.  Earlier, you determined the amino acid sequence of OXA-M290 (MRVLALSAVFLVASIIGMPAVAKEWQENKSWNAHFTEHKSQGVVVLWNENKQQGFTNNLKRANQAFLPASSAKIPNSLIALDLGVVKDEHQVFKWDGQTRDIATWNRDHNLITAMKYSVVPVYQEFARQIGEARMSKMLHAFDYGNEDISGNVDSFWLDGGIRISATEQISFLRKLYHNKLHVSERSQRIVKQAMLTEANGDYIIRAKTGYDTKIGWWVGWVELDDNVWFFAMNMDMPTSDGLGLRQAITKEVLKQEKIIP). Based on the amino acid sequence, what is the molecular weight of this protein? You can use the free ProtParam tool (https://web.expasy.org/protparam/) to calculate the molecular weights of proteins. Make sure to include units in your answer.   Note: The amino acid sequence reported earlier does not include the His-tag that was added to OXA-M290 by the pET-28a vector. However, you do not need to consider the amino acids in the His-tag in your answer to this question.  For Context ONLY: For…

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…

Wh are doing this procedures can you explain? (ex. heating or adding chemicals etc.) 1) Purification of Vitelline from Egg Yolk-Experimental ProcedureMeasure the volume of 3 egg yolks and mix by adding an equal volume of NaCl solution.Extract the mixture with 3-fold volumes of ether and separate the aqueous phase.Do the same procedure 3 times.Mix the sample with water and rinse.It is expected for the protein to collapse.Some more water is added in order to check whether the collapse occurs completely.The sample is centrifuged and the precipitate is dried. 2) Purification of Plasma Albumin and Fibrinogen-Experimental ProcedureAdd ammonium sulfate to 10 ml plasma up to 25% saturation.Separate the collapsed fibrinogen by centrifuge.Increase the saturation level to 33% by adding (NH4) 2SO4.Separate the globulin by centrifugation.Separate prodoglobulins by increasing the saturation level to 46%.Increase the saturation level up to 64% to precipitate albumin.Separate the albumin by centrifuge…