I would like to reuse my primary antibody for western blotting. I am looking for an optimal way to store. I block my membranes in 5% skim milk followed by washing and primary antibody incubation.
can I centrifuge this primary antibody solution and store? Or centrifugation at this point can cause any damage to my antibody
Primary antibody is quite often recycled for WB. However I fail to see where the centrifugation step would come of use.
Usually one just transfert the primary Ab solution from the plastic bag or falcon one used. Or maybe you want to be sure to get every single drop back? In wich case you can indeed centrifuge the tube AFTER transfering the solution in the storage tube at a slow speed for a min or 2, that way even if you damage the antibody, it would be a very small amount. Personally I would just discard it.
Of note, in my experiments, storing the primary AB in 5% BSA instead of skimmed milked increased its durability.
Hello Sulail Fatima,
If you plan to reuse the diluted primary antibody, centrifugation is not essential. The medium used to dilute the primary antibody is important. If you need to reuse your primary antibody, you can make up the dilution in 5% BSA solution. BSA functions as a protein stabilizer, allowing you to reuse the primary antibody longer when stored in the lab fridge at 4 deg C.
But if you want to reuse your primary antibody over an indefinite period of time between blots, you can make up the dilution in 5% skim milk, without sodium azide, and freeze it at -20°C as BSA is not as effective for protecting the antibody from the freezing process as milk is. To avoid repeated freeze-thaw cycles, you may store the diluted primary antibody in low volume aliquots.
Best.
Malcolm Nobre and Henri Weidmann
My antibody diluent is from licor (Intercept® T20 (PBS) Antibody Diluent). I should have added that to my post.
Why I thought of centrifugation was only because upon collection of primary antibody from the blot I saw the solution was not clear. Even though after blocking with milk I washed my blots before adding primary Ab, my observation that the diluent (with Ab) is not clear after I collect it in falcon made me wonder if I can centrifuge before storing at 4 degree for another use.
I observed that my old diluted Ab (used once but not centrifuged) in refrigerator has a pellet at the bottom of the falcon tube which is clearly visible with naked eyes. While the one I centrifuged and kept has minimum to no pellet settled at the bottom.
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