I am working on a dehydrogenase enzyme. I wanted to check the cleavage of His-tag from my enzyme using Western Blot. When I do the Western blot in Phosphate buffer I can't even see that in the positive control (i.e protein with intact His-tag). But the positive control shows clear band when Tris buffer is used as a lysis buffer. I used to store the protein in phosphate buffer. My question is :
Why Tris shows detection but not Phosphate buffer ?
Dear Prem, You had mentioned that you had got the result when perform the western blot using tris buffer, but you were unable to judge the presence of his tag in western blot using phosphate buffer. In both the case you had use the same primary and seconadary antibody right? Your secondary antibody is enzyme linked right? then at first check the stability of the enzyme in phosphate buffer.it may be the case that the enzyme may loose its activity in presence of high molar concentration of phosphate buffer.Moreover the antibody stability in the said phosphate buffer may be lower than the tris buffer.
I agree with Hua Xiao: I don't bother performing Western blots to check for tag removal. I run an SDS-PAGE gel with the processed sample, purified or not, side by side with the untreated protein. Even with a small tag like polyhistidine, the size difference between the two samples (~ 2-3 kDa) is sufficient to be detected unless the polypeptide is very large.
What is your blocking reagent? Skim milk? If so, the calcium may react with the phosphate fom your PBS and precipitate on the membrane making the transfered protein inaccessible to your antibody.
I don't understand at which point of the experiment you use the different buffers: At lysis? As dilution buffer for the antibodies? For a Western Blot you typically have a SDS-PAGE with an sample buffer as the first step and a TRIS-based transfer buffer.
Nevertheless I guess it might be a matter of pH. At which pH do you use the phosphate buffer and the TRIS buffer respectively?
Did you make sure that you protein is transferred to the membrane in both cases by staining the membrane with Ponceau S or similar dyes?
As Vivica mentioned, the question is not clear about the stage in which different buffers were used. I suspect that indeed the difference was not in the Western Blot but in the lysis and storage buffer (as one can understand from the question, but not from the title). In this case, it could be that in phosphate buffer your protein was degraded, either by a protease in the lysate or by a microbial enzyme. Phosphate buffers tend to get contaminated with microorganisms faster than Tris.
Which system do you use to detect, Chemilumi or ALP colorization ?
ALP is interfered by inorganic phosphate.
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