Hi I am blotting for p53 in my experiment. The predicted MW is 53 kDa. However the actual MW appears to be higher than the expected MW in SDS-PAGE. I loaded 10 micro-gram protein. I didn't have positive control for p53. I wonder what might happen?
Thanks in advance
Hi Nghiem,
The reasons can be multiple. Usually, if the protein is not fully denatured it can migrate slower. However I would first ask what expression system you are using as proteins expressed in mammalian cells can carry PTM modifications which will artificially increase the Mw of the protein. What is the source organism of the p53 target?
SDS-PAGE conditions, such as the % of polyacrylamide gel and buffer system may also have an effect on the rate of protein migration. Gels with higher % will slow down the proteins, however I would not expect a protein of 53 kDa to be a major issue. You may try a different loading dye / increase the amount of DTT/ BME in your loading dye as a first step.
It is always best to include a positive control to ensure that your primary antibody is specific and the band you observe is genuine.
Hi Nghiem,
The reasons can be multiple. Usually, if the protein is not fully denatured it can migrate slower. However I would first ask what expression system you are using as proteins expressed in mammalian cells can carry PTM modifications which will artificially increase the Mw of the protein. What is the source organism of the p53 target?
SDS-PAGE conditions, such as the % of polyacrylamide gel and buffer system may also have an effect on the rate of protein migration. Gels with higher % will slow down the proteins, however I would not expect a protein of 53 kDa to be a major issue. You may try a different loading dye / increase the amount of DTT/ BME in your loading dye as a first step.
It is always best to include a positive control to ensure that your primary antibody is specific and the band you observe is genuine.
Hello Nghiem Le. As you have mentioned, 53 kDa is only a predicted MW. It is likely that p53 has different MWs from one species to another. It has been reported that p53 has multiple isomers with different MWs (see papers as attached). In addition, I also agree with Dominika that the SDS-PAGE conditions may also affect the protein migration.
Article P53 and its isoforms in cancer
Article The p53 Protein is an Unusually Shaped Tetramer that Binds D...
The former answers have said it all. PTMs, PA concentration etc. To be sure if your antibody is recognizing the right protein you could check via a 2D Blot. Cut out the spots that are recognized by the antibody and check with MS. In a 1D blot there are several proteins in one band so MS identification might be very difficult. With a 2D Blot you can even see, if your protein of interest has different isoforms and PTMs. One 2D Blot can provide some very interesting data.
I agree with all the wonderful answers, comments and suggestions. In addition, I want to raise some points for you to think about them. Is your protein from the cell lysate/tissue homogenate or purified protein? Do you think your samples contain the right amount of p53 that can be detected by your Abs? How many times did you perform the experiment? Did you use the right Ab that can recognize p53? As it is suggested above, it is good to perform MS analysis of the detected band.
Good luck!
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