I'm currently working on a retrovirus. I'm having trouble detecting protein using IP-WB methods.
I have the antibody-it's polyclonal, but I don't have antibodies conjugated with beads for the IP process. Is there a kit available for directly conjugating antibody and agarose beads? Does anyone have any suggestions? Thank you very much.
What species was the antibody raised in? You can likely use Protein G precoated beads, which bind to IgG.
It was a goat. I've tried using protein G-beads as pre-treatment. But, it causes some unspecific bands, heavy and light chains. I just wonder if it will be easier to have some kit to conjugate directly between them.
You could use NHS activated Sepharose or use (e.g magnetic) beads with carboxyl groups and activate them yourself with EDCxHCl and NHS. This approach immobilizes proteins covalently by binding to amino groups. You might still have antibody fragments on your gel, but less than when using Protein G.
If you have a specific antibody against your protein of interest, you could directly label it, e.g., with Biotin, and then use Streptavidin-HRP for detection in WB. In this way, you won't get signals from the antibody fragments on the gel.
Another option is CNBr-activated Sepharose. While the covalent bond is not quite as strong as those from the approaches suggested by Wolfgang Schechinger, the activated beads, more conveniently in my view, are provided as dry powder, whereas NHS activated beads are suspended in a dry solvent and are more prone to loss of functional groups, esp. after opening. Both approaches will conjugate lysines on the Ab and both will have the same advantages over protein G.
Good point, Nick Gee. When I don't trust the NHS activation (e.g. when an opened bottle of pre-activated resin has been sitting in the fridge for some time), I add a little NHS and EDC, let it react for 15 min and flush away excess reagents with a little ice-cold 1mM HCl.
Some additional points are to be considered here. If you have a crude serum from goat, you usually need protein A or G coated beads to selectively immobilize IgG and not other proteins, such as albumin. We have developed a method to link the antibodies covalently, to avoid leaching of the antibodies in the final elution step: Article Preactivation Crosslinking—An Efficient Method for the Orien...
But be aware, that even in this case, only approx. 1% of the antibodies might be specific to your target. This means that you immobilize 99% of irrelevant IgG. In order to avoid this, the use of monoclonal antibodies or target-affinity-purified polyclonal antibodies would be required. Furthermore, it is important that your antibody solution does not contain any competing amines (such as glycine, other peptides, or proteins), which is usually the case if you try to use a crude serum. A buffer exchange or a Protein A purification would be advisable.
Thank you very much for any suggestions and advice regarding my question. Finally, we found a kit to conjugate the antibody to protein A/G beads. I do hope everything goes well.
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