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protein size in SDS-PAGE - (Jul/23/2015 )

Hello everyone

Recently, I have a trouble with my protein which is detected with western blot. If I come to NCBI and look for protein sequence, I saw that my protein has around 360 aa, familiar 39.6 kDa. However, after loading this protein on SDS-PAGE, I calibrated this protein with marker 250kDa, my protein has size like 50 kDa.

So that, I hope that everyone help me to solve this problem. Thank you.

-Vu Hai Ha-

Not sure what you mean by "calibrated this protein with marker 250kDa".  Are you just running a known 250kDa protein as your only MW standard? Or are you running an entire MW ladder? If not doing the latter, re-run your gel with a MW ladder to get the best estimate for MW.


Assuming you are running a ladder, it is not unusual for proteins to migrate a little faster or slower than the predicted MW. Reasons could be due to incomplete unfolding of the protein, crosslinks, disulfides (if not run under reducing conditions), large patches of hydrophobicity or unusual charge distribution, aggregation in SDS, etc.


Double check the sequencing of the gene to make sure you are expressing what you think you are, and/or cut out gel slice with your protein and send for MS/MS sequencing. These will give you piece of mind that you have what you think you have, so that you can move forward with confidence.


Thank you so much for your suggestion. 

If I calculated the number of amino acid of my protein, I saw that it's weight around 39.6 kDa. However, when I loaded my protein in SDS-PAGE and compare my protein position with 250 kDa ladder, I saw that it was around 50kDa.

My protein belong helix-turn-helix family protein and it's stucture has 10% basic amino acid (R,K,H) and 37% hydrophobic amino acid (V,I,L,F,W,C,R,K,H). Is this answer for my phenomenon which I saw in SDS-PAGE.

-Vu Hai Ha-

It is not possible to predict if a protein will migrate aberrantly...most don't but some on occasion do. In reality a 40kDa protein migrating at 50 kDa is not all that unusual.


Are you running the gel under reducing conditions?  Try both ways. Also try with and without boiling to see if that affects migration. 


As I said before, if all checks out, then just resequence your expression construct to make sure that is perfect (if you are indeed over expressing i). And also send a gel slice for MS sequencing to ensure it is your protein.


I already tried to boil my protein after being treated with loading buffer (contain SDS, mercaptoethanol...). Eventually, I saw that my protein might be aggregated because it was formed precipitate eventhough trying with temperature gradients from 70 to 100 degree. After that, I centrifuged this sample and loaded on SDS-PAGE, however I did not detect target protein. 

Let me say something more about my story. If I compare my target band with primary antibody datasheet which is supported from producer company, their band and my band showed the same size, around 50 kDa.

Can you explain this phenomenon to me.

Thank you so much 

-Vu Hai Ha-