You are running a size exclusion column to purify your 25 kDa protein from a lysatemixture. The void volume for the column is 10 mL, while the elution volume is 50 mL.Theresin is used to separate proteins from 10 kDa up to 100 kDa.After running 75 mL of bufferthrough the column, you stop and run samples on an SDS-PAGE gel on fractions thatshowed absorbance values at 280 nm.On your SDS-PAGE gel, none of lanes shows a bandat 25 kDa. What is the best possible explanation for the results?y. Your elution buffer did not have a high enough salt concentration to eluteyour protein.z.Your protein is not globular so runs at a different molecular weight on theSDS-PAGE gel.aa. You did not run enough buffer through the column to elute your protein.bb. Your protein does not absorb at 280 nm due to no solvent exposed aromaticgroups.

Size exclusion column chromatography: As a function of their respective sizes, molecules are…

Answered: You are running a size exclusion column…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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Explain how gel filtration chromatography works. What type of gel will you used when the protein size is 2500 Da? Explain.
You make 100 ml of a stock solution for mounting sections, that contains 0.5g gelatin. You take 10 ml of this stock solution and add it to 990 ml 0.1 X phosphate buffered saline. The concentration of gelatin in this diluted solution is: 0.5% 0.05% 0.005% 0.0005% 0.00005%
4 mL of 10% TCA solution was added to 1 mL of serum and after mixing, it was waited for two minutes and filtered through non-phosphorus filter paper. 1 mL of the filtrate was taken and 13 mL of distilled water, 4 mL of sulfomolybdic acid and 2 mL of dilute SnCl2 solution were added and mixed, and after waiting for 15 minutes, the absorbances of the obtained solutions against pure water at 520 nm were read. If the function of the calibration graph obtained with 0.5-2.5 mg/mL standard phosphorus solutions is y= 0.245x + 0.107 and the absorbance value of the serum sample is 0.109, what is the amount of phosphorus in the sample?
In Gel filtration chromatography, when will you stop collecting eluents if sample is not colored?
The SDS-PAGE protocol requires 0.5L of running buffer for the gel apparatus. The stock running buffer comes as a 20x concentrate. How should you prepare your running buffer? 50ml 20x stock + 450ml deionized water 450ml 20x stock + 50ml deionized water O 250ml 20x stock + 250ml deionized water 25ml 20x stock + 500ml deionized water 25ml 20x stock + 475ml deionized water 2ml 20x stock + 500ml deionized water O 100ml 20x stock + 400ml deionized water
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In SDS page, the ________ proteins will move through the gel fastest and first, and in size exclusion chromatography, the _________ proteins will elute from the column first. smallest; largest largest; largest largest; smallest smallest; smallest The volume of the beads is included in (Select all that applies) VT Vo Vi VS VL
A pharmacist prepares 80 mL of an isotonic solution of 1.1 %w/v Lidocaine HCl (E-value 0.2). To prepare the solution, he dissolves Lidocaine HCl in purified water to make an isotonic solution. Then he dilutes the Lidocaine HCl solution with 0.9%w/v sterile sodium chloride to complete the formulation. How many milliliters of purified water is needed to make the formulation by this method?
You have a sample at 50 ng/ul and you would like to load 400ng of this sample on a lane of an agarose gel. You also have TE buffer as diluent and 6x loading dye. Your total sample should be 12ul. Calculate the amounts of each reagent necessary to prepare this sample for gel loading.
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Protein isolation and Purification Technique Isolation techniques Centrifugation Salting out Isoelectric precipitation Chromatographic techniques Gel filtration lon exchange Affinity HPLC SPE Electrophoretic techniques Native PAGE SDS-PAGE Tricine-SDS-PAGE Isoelectric focusing Western blotting Principle of separation
Image 1 shows raw data of gel electrophoresis. Label/annotate image 1 (lanes, ladder sizes, etc). Explain what your seeing in the gel. Use the following information and picture 2 to assist in labelling. The purpose of this gel electrophorsis is to ensure that your GOI, FAP257, is in each BAC. Protocol that was done for Gel electrophoresis: -Place tray with gel into gel box -Fill gel box with IX TAE until the gel is completely submerged -Remove the comb for wells -Load 10ul of the 1kb Gene Ruler ladder (well 1) -Contains DNA ladder, 6X TriTrack DNA, Loading Dye, and Deionized water -Load other wells -Well 2: Control -Well 3: BAC- 15M5 -Well 4: BAC-39K10 -Well 5: BAC-27N17 -Run the gel at 100V for 30 minutes or until the dye front has migrated 2/3 down the gel

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