Hello Marlene Mayer

You have not provided detailed information about your experiment. Nevertheless, I can share some information that could be helpful to solve your problem.

1. Choosing the right gel is a key factor in the successful transfer of high molecular weight proteins. Proteins >200 kDa are compacted into a very narrow region at the top of the running portion of the gel, leading to poor resolution of protein bands. I recommend that you use Tris-Acetate gels which maintain a pH of around 7, high molecular weight proteins can migrate further through the gel, allowing increased distance between protein bands thereby separating out large molecular weight proteins with higher resolution. This will allow better transfer of high molecular weight proteins out of the gel leading to increased transfer efficiencies and higher sensitivity.

You may even use a low percentage SDS-PAGE gel, such as 10% stacking gel and 5% resolving gel. The 5% gel is quite fragile, so you need to be very delicate when handling it. Large proteins will move out of a lower percentage gel much more efficiently than out of a higher percentage gel, and they will also resolve better.

2. The next factor which is critical is the composition of your transfer buffer. Large proteins can precipitate out in the presence of methanol. Avoid this by decreasing the methanol percentage (10% or less) in your transfer buffer.

Larger proteins can precipitate in the gel, inhibiting their transfer. The SDS added during SDS-PAGE usually takes care of this problem, but larger proteins might require some more SDS. So, you may add up to 0.1% SDS in your transfer buffer to discourage precipitation. However, SDS can inhibit binding of proteins to membranes. So, try starting with a lower concentration of SDS (for instance, 0.0375%).

3. The type of membrane used for transfer is another important factor. PVDF membrane for transfer will be more suitable for large molecular weight proteins. Large proteins can precipitate out in the presence of methanol, and PVDF membrane does not require any methanol in the transfer buffer. So, you have a higher chance of successfully transferring your protein to the blot using PVDF membrane. But please do not forget to activate the PVDF membrane with methanol before using it.

4. Another point to be kept in mind is the electrophoretic transfer. It can be done in either semi-dry or wet conditions. Wet conditions are usually more reliable as it is less likely to dry out the gel and is preferred for larger proteins. Getting a good transfer efficiency with large proteins might take a little bit more time. It can be hard to move those big molecules around quickly. Try transferring overnight at 4 degree C at 40mA, but at the same time keeping your transfer cool is also important.

Additionally, I am attaching a paper that will be helpful for your experiment.

Article Tris–Acetate Polyacrylamide Gradient Gels for the Simultaneo...

Good Luck!

There are several potential reasons why you might not be able to detect your protein on a Western blot. Some possible issues to consider include:

To troubleshoot these issues, you can try the following approaches:

• Check the protein concentration and quality.
• Make sure you are using the correct primary and secondary antibodies.
• Optimize the transfer conditions.
• Experiment with different blocking solutions and times.
• Check for non-specific binding of the primary and secondary antibodies.
• Consider using a different membrane or detection system.

I hope this helps! If you are still having difficulty detecting your protein on the Western blot, it may be helpful to consult with a research scientist or a technical specialist at your institution.

How was the housekeeping/reference proteins as well as the ladder?