Hi everyone,
I wanted to compare 2 protein standards (from the same company) to decide which one to use but after I found that the location of molecular weights of the two different standards does not match when I run my SDS-PAGE gel. For example, one standards is supposed to have a protein band with 20 kda and the other one 21 kda; however, the one with the 21 kda band appears far lower comparing to the standard with the 20 kda band. I know that the distribution of the protein bands of each standard is different but shouldnt the molecular weight of both be somehow aligned?
I am attaching a file with images of my gel, in one image I took one standard as basis first and in the second image I am taking the other one.
Thank you in advance for your answers :)
Veronica
Use any pre-stained marker to compare, calculate protein band in Kd according to the manufacture's datasheet provided, run each standard in separate lanes like 2, 4, 6, 8 and leave empty for first and last lane, then check the results.
How the proteins resolve is also affected by their charge, prestaining (if they are pre-stained standards), reduction state and so on. Although the manufacturer might give the theoretical mW of the protein, it may resolve somewhat differently under different buffer conditions, different gel % and so on, just as your own proteins may run at somewhat different apparent mW when compared with defined standards with respect to their predicted values.
Hello
I think you should do 2 things: 1) Please send an email containing your pictures and exact protocol to to the producer company. They can help better than others.
2) Read the guide paper carefully. Are you sure about the exact size of protein? check the expire time. Maybe your protein has degraded or something like that. Do you run them one time or more? if you run it one time maybe the gel was not homogen. You should study the troubleshooting of SDS_PAGE. Then run the 2 proteins again at the same gel.
After several freeze-thaw or rough vortex the protein can be damaged. The band is sharp or has some smear? If it has some smear it is damaged. If the degraded pieces are very small they can not be seen in the gel.
be successful
The determination of the molecular mass of a protein by SDS-PAGE is a pi-times-tumb method that gives, at best, only approximate results. In theory, proteins bind on average 1 SDS molecule per 3 amino acids, which results in a constant charge-to-weight ratio and hence acceleration in an electrical field. At the same time, denaturation leads to similar structures for all proteins. Thus, the Rf-value is determined by the interaction of the protein with the mesh of the gel, and hence protein size. However,
- hydrophobic proteins bind more SDS than they should, so transmembrane proteins run faster than expected. For example, the alpha-subunit of Na/K-ATPase has a mass os 112 kDa, but an Rf-value corresponding to 87 kDa (an error of 22%).
- highly charged proteins like histones also give erronous results.
- some proteins are not unfolded in SDS and hence are more compact, thus run faster.
You can reduce the problem by running Fearguson-plots (Rf value vs gel concentration, http://www.his.com/~djt/FergusonKA-Metabolism-October-1964.pdf). But in the end, PAGE is not a first principles method for mass determination of macromolecules.
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