Wavy bands near the bottom of the gel may be due to the presence of a large amount of lipids, which run at the bottom of the gel but not as discrete bands. Loading less protein on the gel may help reduce the waviness. You certainly don't need so much if you are going to do a Western blot but can see a heavy band by Ponceau staining. Try running one-tenth as much.

Adam B Shapiro it’s a tissue lysate and my target proteins are not very abundant in healthy state (control). I am afraid if I see no bands for my healthy tissues how would I quantify that?

is there a way to remove fat contamination from the samples before doing protein estimation? My tissues do have varying amount of fat since it is embedded in fat.

You can use trichloroacetic acid or acetone to precipitate the proteins from samples, wash the pellets with acetone to remove lipids, then redissolve the proteins in SDS-PAGE sample buffer.

https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0049-Acetone-precipitation.pdf

https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/protein-lysis-and-extraction/precipitation-procedures

Adam B Shapiro thank you for sharing these methods for protein precipitation. I have a few questions about how would I proceed with bca assay once I have a precipitate dissolved in small volume of water.

I was thinking to make a small alliquote like taking 0.1ul and bring it to 2ul (the amount I need for bca assay) and remaining protein solution I leave undiluted until I get protein estimation. Once I have numbers I can bring my conc to 3 or so ug/ul but again what will be my diluent? Ripa since I use it for extraction? If so will I be introducing salt and detergents back to my sample.

I usually use 4x laemmli for preparing my samples.

Generally, the precipitates will not dissolve in just water. They will most likely dissolve in 1% SDS with Tris buffer. They should dissolve in SDS-PAGE sample buffer. There is a limit to how much of the precipitate will dissolve, so if the pellets are large, you will have to take them up in a larger volume. If you dissolve the pellets in standard 4X Laemmli sample buffer, you will have a problem with the BCA assay because of the reducing agent and dye, so I'd suggest preparing a 4X sample buffer without those two ingredients. They can be added after the assay.

Yes, the concentration of the loaded sample can significantly affect the resolution of small molecular weight proteins on a Western blot. If the sample concentration is too high, proteins may clump or aggregate, leading to distorted bands or smearing on the gel, particularly for smaller proteins that migrate faster and are more prone to diffusion effects. Conversely, loading a sample with too low a concentration may result in faint or undetectable bands, compromising the accuracy of protein quantification. Optimal sample concentration ensures proper separation and resolution, allowing for distinct visualization of small molecular weight proteins. It is recommended to perform a dilution series to determine the appropriate sample concentration for the Western blot protocol (Mahmood & Yang, 2012).

Reference: Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. North American Journal of Medical Sciences, 4(9), 429-434. doi:10.4103/1947-2714.100998