I want to use western blot to check the protein level of p27, I use the protein ladder from Thermo Fisher. and the p27 antibody from cell signalling. I cut the membrane between 55kDa to 35kDa according to my protein ladder. I got my result as a strong bands higher than 35kDa and a weak bands between 35kDa to 25kDa. My question is which bands is p27? The stronger one or the weaker one? And how can I improve my western to get nice bands and clear result?
Thank you very much!
Dear Jun,
The sample preparation conditions look fine to me. However, after digging some literature, I found out that the higher molecular weight bands that you got positive for p27 are ubiquitinated forms of the same. in your case, you have less free p27 and more ubiquitinated p27. Please find the attached article. I hope this information is helpful to you. Goodluck
The P27 should be between 25 and 30 kDa on the gel. But I have seen in many example blots for P27, there are strong signals at the higher molecular weight. I guess, the protein sample was not denatured properly. But I am not sure. Did you use SDS and DTT or BME in your sample buffer and boiled properly?
Dear Jun,
The sample preparation conditions look fine to me. However, after digging some literature, I found out that the higher molecular weight bands that you got positive for p27 are ubiquitinated forms of the same. in your case, you have less free p27 and more ubiquitinated p27. Please find the attached article. I hope this information is helpful to you. Goodluck
Any type of covalent modification, like ubiquitination or phosphorylation, can affect the apparent size of a fully-denatured protein.
If you are unsure about the antibody, do an siRNA experiment. That should solve your problem and it makes sure (for a later publication), that you (and the scientific world out there) believes that it is really p27 and its modified versions. :)
Oh, and I am not sure..: is this the marker on the left and right? I am not sure how you define a certain band. It looks like it is degraded. Try a new one.
I don't have an idea if p27 is a glycosylated one. If so, I suggest not to heat the samples at 95 degree. Heating generates cross links with other glycated proteins giving rise to high molecular weight products. I had this experience with one of the proteins I worked with. I just used to heat till 45 degrees for 10 min.
Dear Teresa Otto; Thanks for your answer.The marker is new and I have them in both side. The left side I add 3ul ladder and the right side I add 1ul. And I define the position of the protein marker according to the memberane also. The attachment is how the memberane looks like. So it should be correct. Acctually there are small white line if you notice carefully where I think is the exact position of protein marker. I think it's white because the markers are too strong.
Ah okay. Still... siRNA experiment is important if you can't judge which band is the correct one. Every cell is different and might show different modifications or unspecific bands. I am sorry to say. :) Good luck Jun
In our experience p27 comes at 25kdsize band (BioRad marker). You may have to run little longer for the separation. Also it appears that the band appears at or on the cut of the membrane. If you want to be sure you can use ab from other companies. We use both Cell signaling as well as Santa Cruz. Both worked.
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