Hello Elisa Adani

While working with low MW proteins, you will need to take care at the protein transfer stage. Low MW proteins are susceptible to over transfer, loss of sample due to lack of retention by the transfer membrane and/or too rapid transfer.

For low molecular weight proteins (MW< 20 kDa), 0.2 µm PVDF membrane may be used. 0.2μm can prevent proteins from directly passing through the membrane during transfer. Soak the PVDF membrane in methanol for 1-2 mins to activate the positive group and facilitate binding with negatively charged proteins. Then, put the PVDF membrane in transfer buffer.

For small proteins, less is often more when it comes to transfer length and voltage (or amperage depending on your setup). How much you should adjust these will be brand and model system-specific. Blotting systems typically come with recommendations for current and transfer time. I would recommend that you should refer to the equipment manual or website for the transfer time and voltage for low MW proteins. Generally, 200mA constant current for 30mins for proteins of MW less than 10kDa is used, in case you are unable to find this information from other sources.

Another important point you could consider is soaking your gel in an SDS-free buffer (or simply water) for 5 minutes prior to setting up your transfer. This helps to remove SDS, which coats small proteins in negative charges, increasing their rate of passage through the western blot membrane.

Remove SDS from transfer buffer. All proteins are hindered from binding to membrane by SDS, especially for small proteins. Keep the methanol content at 20%. This will help remove as much SDS as possible and improve transfer efficiency.

Transfer buffer for wet transfer

25 mM Tris base

190 mM glycine

20% methanol

Check the pH and adjust to 8.3

For transfer of small peptides like 1-2 kDa, a shorter transfer time and lower voltage should be used to prevent loss of peptides and minimize smearing. Here are some suggestions for blotting buffer, time, and voltage:

It's also important to ensure that the PVDF membrane is properly activated and wetted before transfer. This can be achieved by soaking the membrane in methanol for a few seconds, followed by washing in water or transfer buffer.

Overall, the key for successful transfer of small peptides is to optimize transfer conditions, such as buffer composition, transfer time, and voltage, to achieve the best possible transfer efficiency without losing or smearing the peptides.

In addition to the suggestions above, you may use PVDF membrane as a double fold in order to verify transfer time or voltage and to catch your peptide if any over-transfer issue exists.

You may add a tag to your peptide to increase its MW if transfer and blotting are not applicable in any case...