Dear Jaleel Miyan

if the samples were stored at -20°C, you can use it.

To be sure thatr sample is still reduced you can add some more reducing agent and repeat the heat cicle before load. I do not have a lot of experience with mercapto, but with DTT and/or TCEP the sample are quite stable for long time at -20°C.

best regards

Manuele

Thanks Manuele, that's very helpful.

Hi Jaleel,

It is alright to store protein samples in Laemmli buffer supplemented with BME in the fridge.

Best,

Shayan

The samples stored frozeb should be ok, just heat them again. In the fridge I'd store them at most o/n, there are proteases that survive boiling in sample buffer.

Thank you Shayan and Engelbert, very useful and helpful. So frozen ok and dont keep in fridge except overnight.

Hello Jaleel,

In my experience, the samples stored at -20°C have a longer shelf life than the ones stored at 4°C. You can still run them on SDS-PAGE gel to confirm the protein's quality. Yes, it is very common practice to store the samples at 4°C overnight. Don't forget to reheat and cool down the samples before loading them on gel. Good luck!

Brilliant, very helpful Kiran

Hello Jaleel,

In my experience, samples that have been heat-treated in Laemmli buffer do not give a nice profile when they are kept at -20°C and reloaded after a few days. I have observed partially degraded proteins and smears in these conditions. When I have to reload samples at different times I keep them in Laemmli but I make aliquots and I heat only the aliquot I want to use. I don't know the reason for the discrepancies with the other answers, maybe it depends on the proteins you're looking at or maybe their samples have not been heated before conservation at -20°C. Some gel/staining systems (precast gels for instance) also make small bands and smears less visible.

To summarize, maybe you will be lucky because your proteins are sturdy and resist that treatment but be careful, maybe do a Ponceau a Coomassie staining to check the general state of your samples and take your results with a grain of salt.

Best,

Olivier

Yes, protein samples in Laemmli buffer after heat treatment can be stored in the freezer for several months without significant degradation. Laemmli buffer contains a high concentration of denaturing agents, such as SDS and urea, which can stabilize proteins and prevent their degradation during storage. The addition of reducing agents, such as β-mercaptoethanol or DTT, further helps to protect proteins from oxidation and degradation.

When you are ready to use the samples for Western blot analysis, it is important to ensure that the samples are completely thawed and mixed thoroughly to ensure that the proteins are in solution. It is also important to confirm the quality and quantity of the protein samples using a protein assay, such as a Bradford or BCA assay, prior to loading onto the gel.

Overall, as long as the protein samples were properly prepared and stored, they should be suitable for protein separation and analysis by Western blot.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

Yes, protein samples in Laemmli buffer after heat treatment can be stored in the freezer for several months without significant degradation. Laemmli buffer contains a high concentration of denaturing agents, such as SDS and urea, which can stabilize proteins and prevent their degradation during storage