Why antibodies (primary and secondary) do not cross react with these protein? What chemistry is there to choose these proteins as blocking reagent?
If the membrane is not blocked, the antibodies would bind to the polymers unspecifically (Mainly van-der-Vaals forces), since it is designed, so that proteins (the ones you blot) can effectively stick to it. Otherwise you would lose your proteins. The blocking agent serves the sole purpose, to unspecifically stick to the membrane, and cover it in all the spaces where no protein is bound, simply to inhibit anymore unspecific binding.
The antibody can hardly bind unspecifically to proteins, other than its target protein, since it has a high affinity for a certain epitope. It’s basically the same reason, why none of the other proteins you blottet should get stained (unless they have a structure very similar to the epitope the antibody binds to). The only chemistry behind this is, that most proteins are not sticking to each other, because of their hydration shell, unless they have some affinity interaction like antibodies do (this is also necessary, since otherwise in the cell all proteins would just clump together).
BSA contains mainly albumin and milk mainly casein. Both are mainly used, because they are readily available and especially for milk because they are cheap. It would of course be possible, that if you cultivate a new antibody, that it has a cross-reaction with these proteins, however commercially available antibodies are of course tested for such a cross-reaction. Furthermore, if an Antibody would cross-react with Albumin, it would likely lead to severe blood clotting, since the Albumins are very similar between species, making it unlikely, to obtain an antibody with noticeable affinity for BSA proteins.
If the membrane is not blocked, the antibodies would bind to the polymers unspecifically (Mainly van-der-Vaals forces), since it is designed, so that proteins (the ones you blot) can effectively stick to it. Otherwise you would lose your proteins. The blocking agent serves the sole purpose, to unspecifically stick to the membrane, and cover it in all the spaces where no protein is bound, simply to inhibit anymore unspecific binding.
The antibody can hardly bind unspecifically to proteins, other than its target protein, since it has a high affinity for a certain epitope. It’s basically the same reason, why none of the other proteins you blottet should get stained (unless they have a structure very similar to the epitope the antibody binds to). The only chemistry behind this is, that most proteins are not sticking to each other, because of their hydration shell, unless they have some affinity interaction like antibodies do (this is also necessary, since otherwise in the cell all proteins would just clump together).
BSA contains mainly albumin and milk mainly casein. Both are mainly used, because they are readily available and especially for milk because they are cheap. It would of course be possible, that if you cultivate a new antibody, that it has a cross-reaction with these proteins, however commercially available antibodies are of course tested for such a cross-reaction. Furthermore, if an Antibody would cross-react with Albumin, it would likely lead to severe blood clotting, since the Albumins are very similar between species, making it unlikely, to obtain an antibody with noticeable affinity for BSA proteins.
Some additions to the previous detailed and useful reply.
1)Serum albumin-crossreactive antibodies are usually obtained, when monospecific antipeptide antibodies are prepared via animal immunization by peptide-BSA conjugates. In this case I usually strongly recommend affinity purification of such antibodies on a chromatographic medium with attached peptide antigens, in order to avoid cross-reactions with serum albumins.
2) Gelatin and casein partial hydrolyzates are sometimes used as blocking agents in Western blotting and ELISA, if an antigen under determination binds BSA and/or casein. However, these are rare cases and usually occur with hydrophobic haptens.
Besides serum albumin and milk powder, it is also possible to use serum, for example whole goat serum. If in this case you use a primary antibody produced in goat, non-specific antibody-binding is largely suppressed.
Hello Saturation of western blot with low cost protein !!!! cordially
Thank you very much all for your kind and valuable feedback. Now it is clear to me.
Regards.
There are many other good blocking agents, I use PVA (polyvynl alcohol) there other things as well. A fish protein blocker and some carbohydrate based blocker. Try them all until you get good results.
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