BSC 2010C Biology I: Analysis of SDS-PAGE Gel

Question:

Lab report 1 Discussion: 

Provide independent thought and analysis of your SDS-PAGE gel. Discuss band locations, intensity, and relative purity of each lane. Are the samples properly normalized? Does the lysate have more impurities than the affinity and IEX samples? Is the purity of GST-GFP increasing after each step, and does this agree with the purification table? Is the intensity of the GST-GFP band increasing after each step? Are any contaminants disappearing after each step? Is any GST-GFP found in the affinity or IEX flow through lanes? Why were GST, GFP, BSA, and lysozyme added? 

Lab Report 2 Discussion: 

Provide independent thought and analysis of your western blot. Discuss band locations and intensity of each lane. Are the samples properly normalized? Is the relative intensity of GST-GFP increasing, decreasing, or consistent? Are GST-GFP, GST, or GFP found in the affinity or !EX flowthrough lanes? Why were GST and GFP added? Are BSA and lysozyme visible? Why were they added? Does this differ than the type of control they were in SDS-PAGE? Are there any nonspecific interactions in the lanes? What might they be, and what can cause this? What steps could be taken to prevent nonspecific interactions (consider the washes, types of antibodies, etc.)? In addition, compare the results from the gel and western blot to the purification table results from Lab 6, and discuss the success of the GST-GFP purification protocol. 

Additionally, analyze the protein crystallization results. Describe the shape and size of the crystals, which reagent concentrations gave the best results, and whether you saw individual crystals, star-shaped or twinned crystals, or precipitation, and possible explanations for this. 

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