Western blot optimization

Working with specific proteins, such as phosphorylated proteins or histones, may require some protocol optimization. Below, we highlight the most important tips for performing a western blot with phosphorylated proteins and histones.

Western blot protocol for phosphorylated proteins

If you're working with post-translational modifications of proteins, such as phosphorylation, it's essential to handle your samples with care and use the right reagents to maintain the structural integrity of your proteins. To keep the proteins in their phosphorylated state, add phosphatase inhibitors to the working stocks of your buffers and always keep samples on ice.

It is also important to block the membrane in BSA and avoid using casein or milk-blocking buffers. Casein is a phosphoprotein present in milk, which causes high background signals. A starting BSA concentration of 5% w/v in TBS-T is suggested; however, the optimal concentration is antibody-dependent and should be empirically determined. When using abcam recombinant antibodies, 5% non-fat dry milk can be a good alternative blocking buffer.

Histone western blot protocol

Histones have a very low molecular weight, usually around 10–20 kDa, and due to their small size, you need to be careful when carrying out your western blot.

Tips for successful western blotting with histones:

• Use a high-percentage gel for a clear resolution of histone proteins.
• Use a nitrocellulose membrane with a pore size of 0.2 µm to ensure the optimal capture of histone proteins.
• We recommend using high-quality BSA in your blocking solutions over dried milk powders. However, the optimal blocking is antibody-dependent and should be empirically determined.
• Always use loading control antibodies to standardize your experiments.