I have a problem with Western Blot of Large Protein (Transmembrane receptor 500kDa). I am using 3-8% Tris-Bis Gel and DTT in my loading buffer. I found the protein could not run down the Gel properly. I was advised that not boiling the sample will help. Is that true?
Large transmembrane proteins, especially those rich in hydrophobic regions, tend to aggregate and precipitate when boiled which prevents them from properly entering and running down the gel.
Large proteins require gentle denaturation to maintain solubility.
Boiling may cause them to misfold or aggregate rather than fully denature.
Heat at 50–60°C for 5–10 minutes instead of boiling (95°C). This partially denatures the protein while keeping it soluble.
Use Urea or SDS for Denaturation: You can entirely omit the boiling step and replace it with addition of 6–8M urea or increase SDS concentration (0.2–0.5%) in the loading buffer.
Try a Stronger Reducing Agent Other Than DTT e.g. TBP or TCEP
Use a Lower Voltage for Running the Gel as this will allow the larger molecules to stack more effectively and help these enter the gel.