10 replies to this topic

[dcch]

Hello all. I'm dealing a protein with expected size of about 50kDa. After i knockdown the protein using RNAi technique, i detected the expression of protein using the primary antibody raised in-house.

For the control lane: a sharp band was detected at about 100kDa. No unspecific band was observed.
For the sample lane: No band/extreme thin band was detected at about about 100kDa. No unspecific band was observed.

The sample should be reduced and denatured, because I have boiled them in Laemmli loading buffer. My experiment on other protein did not face this problem.

The band indirectly showed that it was in dimeric form. However, will this be possible after being boiled?  

Thanks for any suggestion!

[Vini]

hey, i dunno what system u r dealing with but if its a eukaryotic protein, is it not likely that the protein ia actually post-translationally modified??? hence, the higher MW, rather than it being dimerized........just wondering

[dcch]

DRN, on Nov 13 2009, 11:20 PM, said:

hey, i dunno what system u r dealing with but if its a eukaryotic protein, is it not likely that the protein ia actually post-translationally modified??? hence, the higher MW, rather than it being dimerized........just wondering

Thank you. Yup I'm dealing with human kinase. But will post translational modification cause such a big change in protein size?

[Vini]

dcch, on Nov 14 2009, 09:24 PM, said:

DRN, on Nov 13 2009, 11:20 PM, said:

hey, i dunno what system u r dealing with but if its a eukaryotic protein, is it not likely that the protein ia actually post-translationally modified??? hence, the higher MW, rather than it being dimerized........just wondering

Thank you. Yup I'm dealing with human kinase. But will post translational modification cause such a big change in protein size?

well, glycosylation can cause such drastic changes..........but on a kinase, i wonder???? just check if your protein has any such predicted modification motifs. on a kinase, i would expect phosphorylations as predominant PTMs and yeah, you are right........to expect such increase in MW due to phosphorylation seems unlikely.......

what is the other protein that you talked about in your first post, which shows the expected MW???

Edited by DRN, 14 November 2009 - 10:17 PM.

[bob1]

Perhaps the antibody is not specific... just because you got one band does not mean it is specific, even with the knockdown evidence, unless you have done a loading control.  Try a longer exposure to see if there is a fainter specific band.

BTW, I work on a 37 kDa protein that runs at 50 kDa with no post-translational modifications.

Edited by bob1, 15 November 2009 - 04:05 PM.

[Vini]

bob1, on Nov 16 2009, 05:34 AM, said:

Perhaps the antibody is not specific... just because you got one band does not mean it is specific, even with the knockdown evidence, unless you have done a loading control.  Try a longer exposure to see if there is a fainter specific band.

BTW, I work on a 37 kDa protein that runs at 50 kDa with no post-translational modifications.

Yup, thats possible too. I too hv a 13 kDa protein that runs at 27 kDa and; another 27 kDa protein that runs at 7 kDa! Many a times, proteins do show anomalous mobility on gel........

[Prep!]

DRN, on Nov 16 2009, 01:13 PM, said:

bob1, on Nov 16 2009, 05:34 AM, said:

Perhaps the antibody is not specific... just because you got one band does not mean it is specific, even with the knockdown evidence, unless you have done a loading control.  Try a longer exposure to see if there is a fainter specific band.

BTW, I work on a 37 kDa protein that runs at 50 kDa with no post-translational modifications.

Yup, thats possible too. I too hv a 13 kDa protein that runs at 27 kDa and; another 27 kDa protein that runs at 7 kDa! Many a times, proteins do show anomalous mobility on gel........

this anomalous mobility on the gel is mostly fue to the hydrodynamic radius of the particualr protein which in turn effects the mobility (steric hinderences). another example is any pegylated molecule!!!

[mdfenko]

sds-page will give you an approximation of the molecular weight of a protein. the approximation can be affected by factors such as amino acid composition (this can have an effect on the density of sds binding and, hence, mobility).

[dcch]

Thanks all!

I performed a PTM prediction using YinOYang 1.2 (Link) to predict O-beta-GlcNAc and phosphorylation sites. Below is the prediction graph:



.......YYY............G..........G.....Y...................Y..G.................      80
...G............................................................................     160
................................................................................     240
................................................................................     320
................................................................................     400
....................................................



It is obvious that the sites are all located in the N terminal.Prediction of its other isoforms, which showed correct MW in SDS-PAGE does not show any such sites (or the sites are spread throughout the protein, with less than 4 sites). Is this prediction helpful somehow in determining this phenomena? Will O-beta-GlcNAc cause such a big shift of size in SDS-PAGE (expected: ~50kDa, observed: ~100 kDa)

Thanks!

Edited by dcch, 18 November 2009 - 03:50 PM.

[jangcastillo33]

will this be an advantage in solving or predicting the outcome regarding to this matter?

IVA

[TKim]

It is interesting to find same problem to mine.
I've had hard time in Western blot of some protein in human heart homogenates.
Western blot with recombinant protein-coding adenovirus infected cells (rat neonatal cardiomyocytes) showed predicted size (98kDa), but I've never got signal with human heart sample in that size. Instead, signal appeared as 250 kDa.

The other discussion in this forum said SDS binding in certain protein can be a cause of this kind of size-shift.