Protein sample preparation for SDS-PAGE - (Oct/08/2006 )
Can someone explains why sometimes we would choose lysis buffer for protein preparation but mostly people choose SDS reducing buffer? Thanks.
I guess you mean why sometimes people use to lyse cells in non-reducing and sometimes in reducing conditions. You can lyse the cells in non-reducing conditions and you will preserve the structure of your proteins. This is helpful if you want to work with an enzyme (like when you do luciferase assays for example) or you want to study protein-protein interactions. If you use SDS you are denaturing your proteins, therefore they won't have the right conformation to interact with other proteins (and they'll not be able because they are also "surrounded" by a negative charge due to the sulphate groups of teh SDS) or to perform their activity (in the case of teh enzymes).
usage of lysis buffer: to preserve protein activiy (catalytic and/or binding) and to determine protein concentration;
lysis with SDS: mostly as Laemmli buffer to perform SDS-PAGE; direct usage (without non-SDS lysis buffer) makes it difficult to determine protein concentration and as it was said to preserve activity; some use it direct for SDS-PAGE because of low quantity of cells, better than scrapping of cells or dilution in in non-SDS containing lysis buffer
Hi, I have one question: for WB we use Laemmli buffer, so proteins lost their structure. Therefore, how can antibodies recognize the antigen?
Thanks a lot~
umm...do antibodies regonize a part of the protein structure only? e.g. a short chain of peptides or polypeptides?
Hi, I have one question: for WB we use Laemmli buffer, so proteins lost their structure. Therefore, how can antibodies recognize the antigen?
umm...do antibodies regonize a part of the protein structure only? e.g. a short chain of peptides or polypeptides?
On the datasheet of an Ab it is indicated whether the Ab is suitable for detection in WB, IF, for IP, etc. The Ab recognizes a small part of the protein, an epitope. This can be a surface epitope that is accessible to the Ab when the protein is folded or when it is either folded or unfolded; or it can be a region of your protein that is not accessible when the protein is in the native form; it can also be that the Ab recognizes a specific epitope that exists only when the protein is folded and in this case you will not be able to use the Ab for WB
good answere from dnafactory; one addition: there is to distinguish between monoclonal which detect only one epitope, and polyclonal Ab which detect multiple epitopes; so polyclonals are more flexible to use in various applications (Wb, IP, IHC/ICC, ELISA and others) or the other way round: monoclonals may be more critical for different applications; each Ab has to be tested for a distinct method before routine usage
I have one question. I was told to change buffer from Lysis to PBS 1x before determining the concentration of protein before bca because imidazole in lysis buffer interacts with reagants. Is it true?
PBS is no a buffer but buffered saline which means you will not only substitute imidazole but also add salt; a too high ionic strength is generally not to recommend in lysis buffer but this depends on what you like to do with lysate....
imidazole may react to aminoketons which mimic peptide bonds