DSS (or other salt?) precipitates when diluted in PBS (10-fold, DSS in DMSO).
I wouldn't suggest DSS, but rather the water soluble analog BS3 (http://www.proteochem.com/bs3crosslinker100mg-p-39.html) for this application. This link is for an antibody orientation protocol using BS3:
http://www.proteochem.com/protocols/Antibody-Orientation-Protocol.pdf
Its normal. DSS precipitates in aq. solutions. The reaction will proceed anyway (This is what the manufacturer says!).
Thanks for the responses. I did get DSS to work despite the precipitation - "micro precipitate", but as you said, BS3 will be better. I ordered some and will give it a try.
Thanks,
Steve
Currently I am doing similar experiments. Recently I tried to cross link rabbit IgG to protein A using DSS. The cross linking was successful as there was no co elution of antibody. However the overall yield of IP was low. Can anyone suggest way to overcome this problem?
I also met the problem,when I crosslinked the antibody to protein G, the yield of IP is low, dose anyone know the reason ? thx
Crosslinking may affect the structure of your antibody and reduce its ability to capture its specific antigen. This effect is especially prominent when using a monoclonal antibody. I would suggest trying a polyclonal antibody, or combine 2 or more monoclonals.
If the antibody you're using is already polyclonal I would simply increase the starting amount of whole cell lysate if possible!
Make sure you also check that the antibody was succesfully crosslinked by saving the flow-through after incubation of antibody + beads, after incubation with DSS, and also by boiling the beads that are supposed to have antibody attached. You can run these saved samples and probe with secondary antibody to check.
I get the same precipitates when cross linking protein lysates (after lysing cells).
Fold dilution of 200 (DSS) in PBS too.
I wonder if after cross linking and trying to do the IP for my protein if I should spin down the micro precipitate and not use it for bead incubation. It seems my beads(with Antibody) is getting cross linked despite quenching the reaction with Tris-HCl (20mM) and dialyzing out DSS.... I will try to use BS3 too.
Criseyda, The original post was regarding DSS precipitating (or not fully dissolving in aqueous solution). Depending on the concentration, this is normal for it due to its hydrophobic nature.
You may have more success removing DSS precipitate by centrifugation to eliminate possible unintended down stream reactions.
http://www.proteochem.com/dsscrosslinker100mg-p-74.html
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