Thursday 17 May 2012

The acid test for antibody function?




Check out the marker M (Cohn fraction IgG)


Acetate buffer, pH 5.5                                Acetate buffer, pH 5.05
CIM-DEAE                                                                   CIM-EDA
Again, check out marker M.  Why does it look different on the right side? 
Where is the BSA in CIM-EDA on the right? Did they let the gel run out? Or cut the gel off at BSA level? 
So would there be as many bands under IgG in the other gels also if they let the bands resolve better? 
Probably cleanest bands in lanes 1 and 2 for IgG in CIM-DEAE disk with acetate buffer, pH 5.5 (above).  Would the acidic pH affect the antibody's structure/function?  Can this condition be used for monoclonal IgG or IgG from sera after using the technique in J. Chrom B (2010) (Ref: post "Mixed opinions, disturbed flow) to clean up the antibodies?   Is it cost-effective?



25 mM MOPS buffer, pH 6.5                               
                          CIM-DEAE                                                               
Again, check out marker M - still Cohn fraction IgG or mixture of pure IgG and BSA?


20 mM Tris buffer, pH 7.4                                20 mM Tris buffer, pH 8.0
CIM-DEAE                                                                   CIM-EDA
Again, check out marker M.  It wasn't included in the gel for CIM-DEAE so it was cut and pasted from Fig. 2a (the acetate buffer elution gel for the same disk).
M looks even more different in the gel on the right side!
Gel image quality!!!
There's still liquid between the gel and the scanner glass in the left side image!!!

Dhivya, A., Kumar, B., Prasanna, R., Jayaprakash, N., & Vijayalakshmi, M. (2010). Purification of Monoclonal Antibodies from Cell-Culture Supernatant by Use of Anion-Exchange Convective Interaction Media (CIM) Monolithic Columns Chromatographia, 72 (11-12), 1183-1188 DOI:



Dhivya, A.P., Kumar, B.P., Prasanna, R.R., Jayaprakash, N.S. & Vijayalakshmi, M.A. (2010). Purification of Monoclonal Antibodies from Cell-Culture Supernatant by Use of Anion-Exchange Convective Interaction Media (CIM) Monolithic Columns, Chromatographia, 72 (11-12) 1188. DOI: 10.1365/s10337-010-1787-3

2 comments:

  1. Great post! after reading this blog i came to know about monoclonal antibodies.
    Antibody Conjugate

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  2. It's becoming increasingly difficult (for me) to make out exactly how far people will go with certain ideas, given the present circumstances. Given that Goodwin Biotechnology Inc claims to be making conjugates of proteins, including monoclonal antibodies, with various other molecules, I can only conclude that you are being sarcastic. I do hope that if you take anything away, it is not any kind of insight on what to do to express and isolate proteins from these papers. Please do not use acid and urea to elute your proteins, especially antibodies, even if funding agencies and venture capitalists are willing to give you lots of money to do so and even if journal editors are waiting with open arms to publish your results.

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