We are doing western blot and I want to make sure which protocol is the best according to the protein transfer and blotting step. The normal way is to block the membrane and incubate the primary antibodies overnight after completing the transfer step. However, If we do not have enough time which one is the best, let the membrane dry overnight between 2 papers after completing the transfer or let the transfer process take a long time overnight?
Note: I am working on different sizes of proteins. Some of them have large M.W (170 kD) and some of them small (25-45 kD).
If the transfer is already completed and you don't have time to follow with blocking and antibodies, I would put the membrane into TBS-T buffer and proceed the next day. The overnight transfer is also ok but you would need to optimize the parameters - sometimes for the bigger proteins it is even better to transfer overnight with a low current than 1-2h with a high current.
Hello Ahmed Al-Omar
You could put the membrane into TBS-T buffer. Do not proceed with the transfer process overnight. Low MW proteins are susceptible to over transfer leading to loss of sample.
Best.
Western blotting is a technique used to detect specific proteins in a sample. The protocol typically consists of several steps, including protein transfer, blocking, primary antibody incubation, secondary antibody incubation, and detection.
The transfer step is the process of transferring proteins from a gel to a membrane, typically nitrocellulose or PVDF, using an electric current. The efficiency of this step depends on several factors, including the size and charge of the proteins, the gel and membrane used, and the transfer buffer.
If you do not have enough time, it is best to let the transfer process take place overnight. This will ensure that all of the proteins are transferred to the membrane and will also minimize the risk of incomplete transfer. Longer transfer times are typically more efficient, especially for larger proteins.
Once the transfer is complete, the membrane should be blocked to prevent non-specific binding of the primary and secondary antibodies. Blocking can be done with a solution of non-fat dried milk, BSA or other blocking reagents. Blocking overnight is optimal, but if you don't have enough time, you can block for at least 1 hour.
When you have primary antibodies available it is best to incubate them overnight. Incubation for longer periods will increase the chance of detecting your protein of interest. However, if you don't have enough time, you can incubate for at least 2 hours.
It is worth noting that, when working with proteins of different sizes, you may need to adjust the transfer buffer or gel percentage accordingly. Proteins with larger molecular weights (MW) typically require a higher percentage gel or a longer transfer time, while smaller proteins can be transferred using a lower percentage gel or a shorter transfer time.
In summary, if you don't have enough time, it's best to let the transfer process take place overnight, after that block the membrane for at least 1 hour and then incubate with primary antibodies for at least 2 hours. It's important to keep in mind that, for proteins with large M.W, you might need to make some adjustments in the transfer buffer or gel percentage accordingly.
Overnight liquid transfer at low current is typically optimal and should be the gold standard (30 V 16-18 h is typical). Shorter transfer times are OK but need more optimization, especially with contrasting Mwts; your best option might be gradient gels in that case. Drying blots is, in my experience at least, disastrous (but might depend on your blocking/Ab system). Rather, keep membrane wet and block O/N or leave in PBS or equivalent until you're ready to move on. Good luck!
you can let the membrane drys and proceed the next.. day/week/month ... but before western blot just "wet " the membrane 10 secondes in ethanol then in TBS (or PBS) without BSA or milk to ensure a uniform wetting then proceed as usual with "fresh" membrane ...
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