Some of my samples lost low molecular weight proteins after migration and transfer. It happens around 20-40KDA.
I used :
-Ripa buffer with SDS, protease and phosphatase inhibitor cocktail.
-Laemeli buffer + DTT and boiled 5min at 99degree.
Does it look like some residual protease activity in certain samples, with more affinity for low molecular weight proteins ?
On the picture you see 4 times the same samples, so it seems to really be a sample effect, and not a problem with the gel, migration, transfer or ponceau.
You can increase protease inhibitor concentration or switch to a different cocktail, perhaps one more tailored to your specific protein's needs. Check the stability of your target proteins and consider using additional stabilizers during lysis or reducing incubation times.
Reduce sample loading to avoid overloading the gel, which can cause poor transfer and loss of small proteins. Optimize gel concentration to ensure good separation and migration of proteins in the 20-40 kDa range.
Adjust transfer conditions, including increasing transfer time or voltage, or try membrane PVDF.
It may help to run a standard protein ladder or marker on the same gel and track how the low molecular weight proteins migrate and transfer. This could help identify whether the issue is with the gel or transfer step.
Thanks Nicolas Poirier
I will take a closer look at my protease inhibitor cocktail, maybe it was too old. What would be the results of not using proper protease inhibitor cocktail when looking at the ponceau , should it be a smear in the high molecular weight, low molecular weight or just a lost of low molecular weight like in my case ?
Frédéric Bégin , how's your going?
- It may be that low molecular weight proteins are small and can easily penetrate 0.45 μm pore size PVDF or nitrocellulose membranes during transfer, resulting in "loss" It is recommended that you switch to a membrane with a smaller pore size (such as 0.2 μm PVDF membrane), which can effectively retain small molecular weight proteins. And shorten the transfer time to avoid over-transfer. Or use a high concentration gel (such as 12–15% or Tris-Tricine system) to better separate 20–40 kDa proteins.
- If you suspect that the problem is with the sample, we recommend some measures to avoid degradation of low molecular weight proteins. For example, try to shorten the boiling time (such as 95°C, 3 minutes) or use milder denaturation conditions. Also, be sure to use fresh samples.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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