western blotting - (Nov/28/2007 )
QUOTE (MKR @ Nov 30 2007, 03:20 PM)
without knowing the type of transfer you are performing it is hard to figure a good transfer time.
I wouldn't mind a good reply where you gave different samples of transfer time/voltage and the rationale since I am (obviously) not doing the same WB as this person, but would love to understand the transfer better. Like the SDS thing is good to know (I'm assuming it keeps the protein from aggregating or something), for possible future studies.
the sds helps maintain the mobility of the protein in the electric field. if the buffer does not contain some sds then the sds from the gel and bound to the protein will exit the gel prior to the protein. this will compromise the mobility of the protein (more important with large proteins, small proteins and peptides do not require sds to transfer).
but, don't add too much sds because it will interfere with the binding to the membrane.