Let's say we have two protein samples with sample A containing half the amount of a protein of interest as sample B. During blotting (PVDF membrane) a portion of the protein will bind to the membrane, some protein may "blow through" the membrane, and eventually some protein may remain in the gel. As long as the membrane has enough binding capacity, the different amounts in both samples will be represented in the blot, as the protein transport rate from the gel into the membrane, as well as the rate of protein leaving the membrane should both be constant. But what happens if the protein amount in both samples is higher than the binding capacity of the membrane? Will sample B reach the capacity limit and then just pass through without binding while more and more protein is bound from sample A, until it also reaches saturation (the result only reflects binding capacity of the membrane)? Or will excessive protein bind to a larger membrane area as soon as the capacity is reached (differences between samples will be represented in the blot)?
my answer to your question is YES.
For each and every experiments optimization is an essential step for obtaining good and accurate data. As far your question is concerned, you have to confirm what quantity of sample you are loading and how much quantity of your sample A and Sample B is in there. after that you have to compare the binding properties of PVDF membranes, and choose the best membrane and best sample volume. and perform WB.
Inorder to confirm the accuracy, do a densitometric analysis. In your querry you stated that "sample A containing half the amount of a protein of interest as sample B", so if the same result you obtained after your densitometric analysis, your experiment is success and thats the best suitable condition for your samples.
for PVDF membrane binding properties, check this link
http://www.scie-plas.com/documents/SCIE-PLAS/distributors/450.pdf
Hi, I am not sure about that, but if you are worried that protein of interest might pass the membrane try to optimize the transfer time, specially if your target is low molecular weight, you need to reduce the transfer time.
my answer to your question is YES.
For each and every experiments optimization is an essential step for obtaining good and accurate data. As far your question is concerned, you have to confirm what quantity of sample you are loading and how much quantity of your sample A and Sample B is in there. after that you have to compare the binding properties of PVDF membranes, and choose the best membrane and best sample volume. and perform WB.
Inorder to confirm the accuracy, do a densitometric analysis. In your querry you stated that "sample A containing half the amount of a protein of interest as sample B", so if the same result you obtained after your densitometric analysis, your experiment is success and thats the best suitable condition for your samples.
for PVDF membrane binding properties, check this link
http://www.scie-plas.com/documents/SCIE-PLAS/distributors/450.pdf
Agree with Pramod, BUT I would not wonder to much from protein from extract using reasonable amount of protein (5 to 50 µg).
For recombinant protein, the question is more complicated
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