Hello all!
I have completed a test yeast expression for 5 different proteins, and I have taken a sample of the culture at 0, 24, 48 and 72 hours. 3 of my yeast cultures seemingly expressed a protein (through SDS-PAGE analysis of the the supernatent from each sample), through the appearence of a protein band over the 4 time points. However, the protein bands all appear at almost the exact same size (Approximately 11 - 12kDa), even though they are all different sizes, 8kDa, 11kDa and 18kDa.
My inital thought was that I cross comtaminated some of my cultures, and they are expressing the same protein...however,
1. they each have a different expression pattern and intensity,
2. I completed tranformation and growth in the same way for each, so why did the two cultures not express anything?
3. I completed an additional transformation for one of my colleagues proteins at the same time, and their protein appears at the expected size (around 14-15 kDa), so my technique was not incorrect.
I am currently completing a western blot, probing for the 6x his tag. However, I was wondering if anyone has any thoughts on why 3 of my 'expressed' proteins are appearing as the same molecular weight, what is the most likely explanation of this?
Thank you!
Have you checked your apparatus? If the electrodes on your gel tank are faulty there can be an uneven electric field. Try loading your samples in the reverse order and see if the problem persists.
Dear JAmes
which % of gel are you using? Your protein are quite small, is possiblle that the resoution of your gel (if you are suing a low acrilammde % (eg 4-12%) is not enough to well differenziate it.
Are your protein expressed in E.coli or in mammalian?
best regards
Manuele
Manuele Martinelli Thanks for your reply, I am using 4-12% and my proteins are expressed in yeast. I have attached pictures of the five proteins.
Predicted molecular weight:
JR2: 15 kDa
JR3: 9 kDa
JR4: 11 kDa
I've also attached a western blot where we probed for 6x his tag. Size marker, JR1, JR2, JR3, JR3 (2), JR4, JR5. You can see that only JR2, JR3 and JR4 was detected with his tag.
It will be difficult to distinguish those 3 molecular weights given how close they all are to each other, even when run at a 12% gel range; I would at least start with a 15% gel (non-gradient) to see if you can get a little better resolution.
Hello James Rooney
By referring to the expected molecular weights, I would recommend a higher gel percentage, around 15% would give you a better resolution.
Best wishes!
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