Affinity chromatography protein Purification
Guidelines for the Affinity Purification Lab Report (lab report 4, to be written individually)
1) Estimate the molecular weights (using Rf values) of the three prominent bands
you see in your thrombin digest lane.
2) You have been provided with two results of a MALDI-TOF analysis (pp. 102-103
above).
a) Calculate the actual molecular weight of the three bands by doing a ratio calculation, based on the observed MALDI MW of the hemoglobin standard and the actual MW of the hemoglobin standard. Ratio = observed value/actual value.
b) What do you think the 33941.3 peak on p. 102 corresponds to?
c) Compare the molecular weights obtained for your three prominent bands
by using Rf values to the MW obtained by MALDI-TOF. How much discrepancy (if any) did you see between the two methods? If you wanted to be as accurate as possible in obtaining the MW of a protein or peptide, which method would you use and why?
3) In developing this lab, we had to make a decision on what protease to use to
separate our protein of interest from the GST tag. We had a number of proteases
to choose from. Copy and paste the amino acids sequence provided (GST+ protein X) into the PeptideCutter tool window (http://ca.expasy.org/tools/peptidecutter/) and look at the following enzymes for your results and answer why you think we chose thrombin over trypsin. Also indicate where thrombin cleaves.
4) Copy and paste the amino acid sequence of the shorter peptide segment
(post-cleavage with thrombin) in Protparam window (http://ca.expasy.org/tools
[protparam.html) to gather the following information:
a) the amino acid composition in percentage
b) the theoretical pl
c) the estimated molecular weight
d) the total number of negatively charged residues
e) the total number of positively charged residues
) the atomic composition (number of CH,N,O,S)
g) total number of atoms
h) the aliphatic index
5) Can you identify this protein using this program? (Use Protein BLAST at the
National Center for Biotechnology Information at https://blast.ncbi.nlm.nih.gov/Blast.cgi
6) Attach all of the obtained data in the Appendices section of your lab report.
Important note on using bioinformatics web-based data in your lab report: you
must provide correct and complete references for the programs that you used to obtain any (and all) data for your lab report.
Complete references for the programs, such as Protparam, Protein BLAST, etc. are provided on the websites these programs run from. It is incorrect to provide an Internet (weblink) reference only.
For example, click on the References/Documentation link in the main ProtParam webpage (located above the data entry window) to view the original references for this program. You will lose marks on your lab report 4 if you do not provide original references for the web-based programs you used to obtain your data. Attach all data obtained to your lab report in the Appendices section.
