Hai. I break the cells containing my expressed protein (r-msAdh1) using non - denaturing lysis buffer. Then I run 10 uL lysate under denaturing polyarylamide gel (SDS-PAGE). But I observed that my protein size is larger than expected (more than 55kD). Expected size is 43kD.
Is it normal? Anyone experience this?
Hi there,
What you should do is actually compare the behaviour of your protein in both conditions (ie. native and denaturing) as sometimes proteins run naturally "bigger" than their actual size on SDS/PAGE.
If the protein has a signal sequence that is normally cleaved by cellular peptidases during maturation and secretion, but the protein is expressed in insoluble form in the cytoplasm, the signal sequence will not be cleaved off, resulting in a larger protein.
Running nondenatured protein on a denaturing gel may result in incomplete denaturation, resulting in anomalous migration. It is not clear from your question whether you prepared the sample for electrophoresis in denaturing sample buffer, however.
It ain't a denaturing gel. However, the increase may be owing to glycosylation or other factors as mentioned by Adam B Shapiro . Also, go for a reducing PAGE.
Adam B Shapiro I prepared the sample for electrophoresis using both of denaturing sample buffer and non denaturing sample buffer. Both of the results is the same; size is larger than expected.
Hi,
You may perform a gel filtration analysis applying MW markers as calibrators. Size exclusion analysis can also say something about expected MWs if you have infrastructure to be applied. But achieving good resolutions and choosing closer mass calibrants to your target protein is essential for accurate results.
SDS-PAGE technique & results could be thought as out of the troubleshooting after conducting this kind of confirmatory analysis, than it may be considered as protein expressional contingency or misevaluation.
Thanks
Adam B Shapiro I have checked my protein using http://www.cbs.dtu.dk/services/SignalP/ , my protein doesn't have protein signal.
Maybe you are getting read-through of the stop codon. It occasionally happens. How large would the protein be if it read through the first stop and got translated to the next one?
Hi Mastura,
I think that your protein is already at the same level of 43 kDa marker line. I count 6 line and that means 43 kDa. I checked the EZ Run Prestained Rec Marker and there are 170 kDa, 130 kDa, 95 kDa, 72 kDa, 55 kDa, 43 kDa, 34 kDa... bands. 6. line is 43 kDa not 55 kDa. Protein markers show different migration patterns in different gel concentrations and they also give different Mw in different gel buffers. If you want to be sure, run a purified 43 kDa protein or more accurately run a commercially available of your protein of interest.
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