**Experiment Description and Results Analysis**You are conducting an experiment involving a size exclusion column to purify a 25 kDa protein from a lysate mixture. The procedure has the following parameters:- The void volume for the column is 10 mL.- The elution volume is 50 mL.- A resin is used to separate proteins ranging from 10 kDa to 100 kDa.After running 75 mL of buffer through the column, samples are taken and analyzed using an SDS-PAGE gel. The analysis focused on fractions showing absorbance at 280 nm. Surprisingly, the SDS-PAGE gel does not reveal a band at 25 kDa. The most plausible explanations include:**Highlighted Possible Explanations:**- **(y)** *Your elution buffer did not have a high enough salt concentration to elute your protein.*- **(aa)** *You did not run enough buffer through the column to elute your protein.***Other Considerations:**- **(z)** Your protein might not be globular and may migrate differently on the SDS-PAGE gel due to its shape.- **(bb)** The protein may not absorb at 280 nm possibly due to a lack of solvent-exposed aromatic groups.This information outlines the steps and considerations involved in troubleshooting issues in protein purification using size exclusion chromatography and SDS-PAGE analysis.

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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Standard curves You are setting up a standard curve for lysozyme. You have a stock vial containing 2 mg/ml of lysozyme and you have 2 ml in the vial. You will want to have a minimum of 1 ml of each concentration for the spectrophotometer. How would you proceed? (Saline is added to make the various concentrations). Concentration Am't stock lysozyme or am't of a previous dilution (note which) Amount saline Total volume wanted 1.00 mg/ml 0.75 mg/ml 0.50 mg/ml 0.375 mg/ml
When run under nondenaturing conditions (without SDS or β-mercaptoethanol), nativepolyacrylamide gel electrophoresis (PAGE) allows proteins to maintain biological activity, so the GFP band should fluoresce when exposed to UV light. How do the staining patterns of gels run in this way differ from the staining patterns of gels run using SDS-PAGE?
Compare and contrast the following protein characterization techniques in terms of the principles governing their functions. electrosrapy ionization mass spectrometry vs polyacrylamide gel electrophoresis
Five dialysis bags, impermeable to sucrose, were filled with various concentrations of sucrose and then placed in separate beakers containing an initial concentration of 0.6 M sucrose solution. At 10 minute intervals, the bags were weighed and the percent change in mass of each bag was graphed. Which line represents the bag that contained a solution isotonic to the 0.6 M solution at the beginning of the experiment? Which line represents the bag with the highest initial concentration of sucrose? Which line or lines represent bags that contain a solution that is still hypertonic at the end of 60 minutes? What is the best explanation for the shape of line e. after 50 minutes?
"If you count 70 colonies from a urine culture obtained using 0.001ml calibrated loop, how many colony forming units (CFU) will be reported?" 70 700 7000 70000 700000
Put the following steps for running a gel in order. first Remove comb and put gel int v [ Choose ] Load samples into gel Let gel solidify for 15-20 minutes Connect electrodes and turn on power to run gel second Pour heated, liquid agarose into gel cartridge Remove comb and put gel into bottom of electrophoresis box Pour buffer into electrophoresis box to cover gel third fourth Pour heated, liquid agarose in v fifth Load samples into gel sixth Connect electrodes and turn v
You perform a Bradford assay. You obtain the absorbance values listed below from the BSA samples; your protein sample yields an absorbance of 1.3; what is the protein concentration of your sample? How did you determine that? BSA (ug/ml) Absorbance @ 595 nm. 25 0.15 50 0.30 75 0.45 100 0.60 150 0.90 200 1.25
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
Assume that you are starting with a sample containing 6 billion active cells. Create a dilution plan to reduce this count to a range of 30-300 colonies on a plate. The powdered sample weighs 500 mg (0.5 g). You can use up to 4 plates – you should plate different dilutions and/or volumes of solution. Start your first dilution by adding 10 mL diluent to 0.5 g powder, which is a 1/20 dilution. Hint: Rearranging the “cfu/ml” formula from our dilutions lab can help you solve this. Choose a target cfu count, and your volume plated should be under 0.5 ml.
The number of bacterial cells in a culture broth is to be determined by a culture technique. Serial dilutions were performed and a 1 mL aliquot from each dilution was mixed with warm molten agar and poured into a Petri dish. The numbers of bacterial colony forming units (CFU) after overnight incubation are shown in the table below. What is the number of colony forming units per mL of the culture broth? Choose only the most appropriate plate for your calculation. Give your answer as the number only (do not add text for the units). You may use scientific notation with the format 1.12e+6 (that is, 1.12 x 106 cfu/mL). (Note: Canvas will then display your answer a whole number.) Plate Dilution Plate 1 10 dilution Plate 2 10 dilution -5 Plate 3 10 dilution Plate 4 10 dilution Plate 5 107 dilution -8 Plate 6 100 dilution *Too many to count Number of colony forming units (CFU) TMTC* TMTC* TMTC* 867 154 18
List two situations when you would not be able to use the UV-Vis absorbance at 280 nm to determine a protein’s concentration.
The number of bacterial cells in a culture broth is to be determined by a culture technique. Serial dilutions were performed and a 0.1 mL aliquot from each dilution was spread onto Plate Count Agar (PCA). The number of bacterial colony forming units (CFU) after overnight incubation are shown as listed in the table below. What is the number of colony forming units per mL of the culture broth? Choose only the most appropriate plate for your calculation. Give your answer as the number only (do not add text for the units). You may use scientific notation with the format 1.12e+6 (that is, 1.12 x 106 cfu/mL). (Note: Canvas will then display your answer a whole number.) Plate Dilution Plate 1 10 dilution Plate 2 10 dilution Plate 3 107 dilution Plate 4 10 dilution Plate 5 107 dilution Plate 6 100 dilution *Too many to count Number of colony forming units (CFU) TMTC* 382 83 10 2 0

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