SDS-PAGE Lab Report: Analyzing rGFP Fractions in BIOL 3380

BIOL 3380 Experiment 6 - Lab Report SDS-PAGE Analysis of rGFP fractions Name: Rania Mohamed Circle your lab section: T–AM W-AM R-AM F-AM T–PM W-PM R-PM F-PM Instructor: Pan Jing Graduate TA: Falah Rahaf Date: 11/01/2023 Partner: Varshini 6- 1

Experiment 6 – Lab Report SDS-PAGE Analysis of rGFP fractions Edit this word document by inserting your answers after each question/space. Please denote your answers by using bold , italic s, or colored font. Do NOT renumber/reorganize the questions. “Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so). Insert graphs (handwritten on graph paper or electronically constructed with horizontal/vertical gridlines) as a clear image with appropriate titles, labeled/scaled axes, and standard deviation bars when appropriate. 1. (4pts) Quality of your SDS-PAGE. You can download the image of your gel from your eLearning lab section. Instructors will evaluate the quality of your gel results. Please see page 6-6 2. (3pts) Construct a figure of your own SDS-PAGE. Include an appropriate figure title, labeled lanes, and labeled ladder bands. You can find an image of the ladder in Lecture 6 notes. MW of ladder bands are 250kD, 150kD, 100kD, 75kD, 50kD, 37kD, 25kD, 20kD, and 15kD. The 15kD band sometimes is not visible on your gel. Please see page 6-6 3. (2pts) What is the estimated molecular weight of your purified rGFP? Show your calculation for credit. Information on how rGFP was cloned in E.coli can be found in Lecture 3 notes. An amino acid has an average molecular weight of 115 Daltons. rGFP has 279 amino acid including epitope tag and Histidine 1aa=115 Daltons so rGFP estimated mw= 279aa*115Da = 32085Da = 32kDa 4. (2pts) Knowing the estimated molecular weight of rGFP, circle/box the band in your E3 lane that is most likely rGFP. *Hint: comparing G0 vs G3 lanes, and wash fractions vs elution fractions will also help you to determine which band is rGFP. Please see page 6-6 5. (1pts) Measure the distance traveled for each band in the MW ladder in the SDS-PAGE figure you create in question 2. Record your measurements in the table below. MW (kD) Distance migrated (cm) 6- 2

250 0.45 150 0.7 100 1.0 75 1.3 50 1.8 37 2.25 25 2.9 20 3.2 6. (4pts) Construct SDS-PAGE standard curve based on data from the table above. Plot the log of the MW versus the distance traveled for each band in the MW ladder. Refer to the lecture notes for an example. Most students will have a bi-phasic curve. Semi-log paper is provided as the last page of this packet. Please see page 6-5 7. (2pts) Based upon your standard curve, what is the relative molecular weight of rGFP? Show your work by drawing your interpolation line on the standard curve. Notice that the estimated and the relative MWs of proteins are not always the same! ~23kD 8. (2pts) What would you estimate the purity of rGFP in the E3 fraction? *Hint: what percentage of the overall intensity (darkness) in that lane is due to the rGFP band? ~30% 9. (3pts) Estimate the total yield (in ug) of rGFP that you collected in E3 fraction. You must show/explain how you calculated your answer. *Hint: base your calculation on your estimated purity of rGFP in the fraction and the total amount of protein present in that fraction (data from lab 5). total amount of protein in E3= 120ug total yield in E3= 120ug*30% = 36ug 10. (2pts) The expression and purification of rGFP started with the growth of the bacterial culture and ended with the elution of rGFP off the Ni +2 -agarose column. Describe one change to this process that you predict would result in an increase in the total yield of rGFP. Increasing the amount of imidazole, would increase the yield of rGFP because imidazole would prevent His-Ni2+ to binding, and more His tagged rGFP would be collected in the elution fractions 6- 3

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11. Eukaryotic Initiation Factor 2 (eIF2) is a heterotrimeric ( , , ) α β γ protein complex that delivers the Met-tRNA i to the AUG start codon during translational initiation. The left panel depicts an SDS-PAGE of purified eIF2 from three different yeast strains that express His 8 tagged versions of a wild-type, a Y142H mutation, or a K250R mutation of the γ subunit. Crude extracts were purified through a Ni +2 -Agarose column followed by a Heparin column. The right panel depicts an equilibrium binding assay of Met-tRNA i to the various purified eIF2 complexes. (adapted from Erickson and Hannig. EMBO. 1996;15(22):6311-6320.) (1pt) Which step in the purification procedure removed contaminating proteins that may contain structural motifs that are similar to a His 8 tag? Heparin column (1pt) What is the estimated purity of the wild-type eIF2? ~30% (1pt) What is the estimated MW of eIF2 γ ? ~55kD (1pt) What is the estimated MW of eIF2? 55+35+40 = 130kD (1pt) Based upon the data, what specific role does the amino acids K250 and Y142 in eIF2 γ function? - K250 increases binding of eIF2 γ to Met-tRNA 6- 4 eIF2[  K250R ] eIF2 eIF2[  Y142H ]

- Y142 almost completely inhibits binding of eIF 2 γ. 6- 5

6- 6

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F23 Lab 6 SDS-PAGE Lab Report (1)

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