I am trying to purify IgM from hybridoma supernatant. I do not need an absolutely pure antibody fraction, a good enrichment is good enough for my work. So, I decided to perform euglobulin precipitation and I dialysed my culture supernatant against distilled water. I successfully obtained a precipitate but I was unsuccessful in dissolving it. I tried Na-Borate buffer at pH 8.5 and PBS (pH: 7.2). Neither were good at dissolving all of my precipitate. Can you suggest any other buffer for dissolving the precipitate or, may I use shear forces like sonication without causing the degradation of IgM? Is it normal that I have an insoluble precipitate?
Hi Özlem,
you need to know the isoelectric point of you monoclonal IgM. If you adjust the pH of your buffer at this pH the molecule is neutral at this pH, there are no charges anymore. If you remove all (as much as possible) salt ions (by using a very thin buffer at 0.001 mmol/L) at this point your protein will precipitate.
Only some pitfalls: You have to run an IEF and identify your IgM (best by IEF + Western blotting). You have to make sure, that you know which one of the bands is the complete molecule. Very often hybridoma producing incomplete immunoglobulin molecules (e.i. not as a pentamer, only heavby chains, not a complete set of light chains, ...). So look also by SDS-PAGE + WB for the molecular weights of your IgM. You can do two immune reactions, one with an anti-µ, the second with an anti-L(Kappa+lambda). If those antibodies are from different species you can use the first anti-species antibody HRP labeled and the second with AP labeled. Run the AP substrate reaction first, the HRP reaction in the second stain.
The key is to identify the IP of the whole IgM molecule.
I'm affraid your precipitate will be difficult to dissolve as you dialised your solution with water. In fact IgM precipitate at low NaCl concentration (below 0.150M). It is better to work at 0.350-0.5M NaCl (too high concentration of NaCl as 2M is also a precipitation factor). You can perhaps try glycine 0.01M known to stabilise IgM .
In crease sodium chloride concentration as suggested by others to 0.15-0.2 M, and what has helped us solubilizing high molecular weight proteins (we worked with phosphosylase kinase) is to add from 5-10% dimethylsulfoxide without any detrimental effect on enzyme activity. Perhaps dialyze the precipitate against a couple of changes of buffer (HEPES, ~pH 7.2) containing 0.15-0.2M NaCl and 5-10% dimethylsulfoxide. Good luck!
Thank you very much for your helpful answers.
I completely agree with Dr. Kiessig and Dr. Karbyshev for pI determination. Actually, now, I am trying to buy ampholites for the old old rotofor in my lab. But currently, with the available items, I will try to isolate my antiboy.
I tried to dissolve the protein in PBS (pH:7.2, 150 mM NaCl), but did not work very well. I will fractionate my preciitate and try higher salt, glycine and DMSO for each bit. Thanks for the advices, I will let you know the result.
Dr. Karbyshev, can you give a reference for PEG6000 pecipitation of IgM. I have PEG 4000, does it also work?
Hello everybody,
I did the experiment long ago but had the chance to write just now.
I tried to dissolve my precipitate in the following solutions and checked for the antibody activity with ELISA, controlled the purity with SDS PAGE and demonstrated the presence of intact antibody with western blot.
1. Borate buffer (pH 8.5) --> small portion dissolved, active, includes intact antibody
2. PBS --> very small portion dissolved, active, includes intact antibody
3. 0.1 M NaHCO buffer containing 0.5 M NaCl, pH 8.3 --> some dissolved, more intact antibody dissolved compared to all others, active
4. 30%DMSO in PBS --> larger portion dissolved, inactive
5. DMSO --> all the rest dissolved, no activity
Thank you for all your help, I think solvent #3 is the best for me since I am planning to conjugate the antibody to cyanogen bromide activated sepharose and that buffer is also used in conjugation studies.
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