Dear all,
We would like to check the purity of a GST-tagged recombinant protein by SDS-PAGE. We purified the protein using Baculovirus expression system.
We run the reducing SDS-PAGE (GST was used as a positive control) to check for purity. But unfortunately, we can only see a small band of our target protein (the expected molecular weight: 65 kDa) except for GST and some cleavages.
I don't know whether it was due to the reducing SDS-PAGE that we used or the protein purification has failed? If it is possible, I hope to receive any of your comments or suggestion.
Thank you,
Yours sincerely,
Tu
actually there is no protein of ur intrest.. but how did you get only GST tag without protein in your purified sample? if protein not purified means no gst tag occurence. but here strange..
1. protein was purified.
2. may be tag cleaved
3. degradation of ur protein
Dear Dr.Konde,
Thank you for stopping by. The protein was newly purified and was thawed only 1 time for SDS-PAGE. So there may be little possibility of degradation of protein, I hope.
So, it could be due to tag cleaved. Do you think it is due to the reducing SDS-PAGE (I included mercaptoethanol)? How can I confirm this one?
im not sure but you can try without reduction after 2 3 times freeze thaw
Dear Tu Trinh,
the weaker band at the same height of your control could potentially have been caused by spillover when loading the wells, I would leave an empty well between the two samples to ensure this is not the case.
Also you are using Coomassie staining which is not very sensitive. To know whether the insect cells (I assume) produced any GTS protein at all, I would do some western blots with an anti GST antibody, using sensitive chemiluminescence.
When checking purity, I would use silver stain not Coomassie. I don't think that reducing buffer will cleave the tag, if this is a conventional protease cleavage tag (e.g. TEV). The reducing buffer will cleave disulfide bonds, not peptide bonds.
I also seem to recognise a weak band around 75 kDa (slightly lower) in your right lane, so it could just be an issue of low sensitivity and spillover?
Hi there,
There is not much visible on your gel. How much of the purified fraction did you load? I would suggest to load much more in order to get a nice band pattern...
Dear Dr.Falcone,
Thank you for your detailing and helpful answer. We tried western blot using anti-GST antibody and we found a relatively small band at around 65 kDa. Therefore, we hopefully got the protein but could not guarantee of the purity.
For the silver staining, may I ask if you know the smallest amount of protein that can be visualized?
Yours sincerely,
Tu
Hi again,
If you have loaded 3µg you were expecting a nice band... So it's either the protein got fully degraded during the sample preparation or/and it doesn't bind coomassie blue properly. BTw could you see the band in the initial cell lysate?
Sensitivity of silver stain is about 1 ng per band or spot, with some kits claiming higher sensitivity. You can stain the same gel with Coomassie first then with silver stain, but usually the molecular weight marker lane will be too intense, as you need to reduce the amounts loaded for silver stained gels
@Dear Dr.Falcone,
Thank you. We will give it a try.
@Dear Liger,
In the initial cell lysate, we haven't tried yet since we wanted to save protein. But as I heard from another lab, they tested the protein already and they just could detect a slight band of our interest protein.
A quick update: the band at around 25 kDa was thought to be polyhedrin, a component of insect cells. Hopefully our protein hasn't got degraded yet.
Tu
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