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Anti-Ryanodine Receptor antibody (ab78378)

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1 question for ab78378

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Question 1

Wednesday 14-December-2011

Blots of mouse muscle homogenate are dark all over, even after stripping and staining for GAPDH.

ANSWER:

  

Thank you for contacting us. I do not see any other complaints in our records for this antibody so it may be that the single vial was defective. I am waiting for protocol advice from the laboratory that validated the antibody for western blotting and will forward that if it differs significantly from what your colleague tried.

One note that we do have recommends using a 10% solution of serum and 1% BSA for the membrane blocking buffer. The serum was from a non-immunized animal of the same species as the secondary antibody host (goat in that instance). If all else fails, there might be an advantage to this over 5% BSA or milk. Stripping a blot and re-blocking with serum may give a better result, but I think it is more likely that there is an issue with the primary antibody.

I am a little concerned that a stripped blot followed by blocking and the GAPDH antibody also gave a dark blot, since the stripping should have removed all the ryanadine receptor antibody, but that is a side issue, more about the efficacy of the stripping protocol than the receptor antibody.

One other thing I meant to suggest was a Ponceau Red stain of the blot before applying the anitbody, to see if high molecular weight proteins transferred effectively. If you need a protocol for this please let me know.

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