Dialysis against PBS of IgG/IgA
Posted 24 April 2009 - 04:20 PM
I've been working trying to put together a protocol for purification of IgA and IgG. I really need a good and complete protocol for purification of these globulins. I'd like to use jacalin for IgA and Protein G for IgG. At the moment, i'd like to know how to recover sample from dialysis tubing (cassette), should i dialyze against PBS Ph 7.2-7.4 for both proteins? Which would be a better buffer to keep precipitated globulins (with amonium sulfate)?
Please, all the information available is welcome.
Posted 27 April 2009 - 05:33 AM
Sounds like you are doing ammonium sulfate precip. I would resuspend pecip in buffer that you will use to dialyze out the ammonium sulfate and would be the matrix buffer used to react with Protein G (on beads? on column?).
Once you have the ab purified store with some azide or freeze -80C.
Posted 04 May 2009 - 11:25 AM
It's true i plan using sulfate ammonium so i guess PBS will be my buffer. However, i found so many protocols online for affinity chromatography for IgA using jacalin but i havent found how to couple jacalin to my matrix. I have some sepharose so i wanted to try the column but i dont even know how to couple/immobilize jacalin (powder) to this matrix. Please send some suggestions to do so, Thanks!!!
Posted 04 May 2009 - 12:47 PM
Another possible method is to purchase a goat/rabbit antihuman IgA and couple this to your matrix. This should work just as well for you and be much more straightforward. I think your goal is to get the purified protein ASAP not to research affinity matrices or conjugation procedures.
As far as conjugation procedures Thermo Fisher / Pierce will provide free technical conjugation help and suggest their products to do the coupling. I found them to be very helpful and knowledgable.