Dear All,
I'm looking for an efficient protocol to purify secreted IgM (Human) from cell culture.
Thank you so much
Selena
Please find the following rapid protocol
Preparation of reagents:
Clarification of the samples to be purified by filtration or centrifugation.
Dissolve saturated sulfate buffer: 750mg (NH4) 2SO4 in 1 liter H2O. Dissolve all salts by heating, then cool down to + 4 ° C. The supernatant is used as the saturated solution
Purification of IgM:
Mixing, in a centrifuge tube, 1ml of supernatant to 1ml of buffer saturated sulfate, quickly but gently mixed by inverting the tube.
let precipitate at 4 ° C for 30 minutes.
Spin at 2500rpm for 30 minutes to pellet the precipitate.
Resuspend the precipitate in 1 ml of 20 mM Tris buffer
Dialyze overnight against the 20 mM Tris buffer, a white precipitate appeared.
Spin at 2500rpm for 10 minutes to pellet the precipitate.
Resuspend the precipitate in 1 ml of PBS buffer to 150 mM.
Read at 280 nm, 1.35 ratio.
attention: IgM is stable in isotonic buffer, do not dilute in hypotonic buffer.
Note: IgG3 can be purified in the same way, avoid mixing IgM-IgG3
hello,
Excuse me, but when I told sulfate buffer, I mean ammonium sulfate buffer.
But I think you'll rectified yourself.
To separate the Ig3 IgM, if necessary, we can modify the protocol, but it doubles the manipulations.
Yes of course....You wrote also the empirical formula in the first answer..
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