It depends on your target protein and needs optimization and experience. However, there are usually 2 types (0.2 um and 0.45 um) for WB and both work well for any size of proteins.

Hello Indhuprakash,

Yes, there are different pore sizes of the nitrocellulose membrane are available. you can select the pose size base on the size of your protein of interest. In general, 0.45 μm pore size is commonly used for proteins greater than 10,000 MW, but 0.1 and 0.2 μm are also available.

Good luck!

Debasish Kumar Dey and Arman Rahimmi Thank you so much for your valuable suggestions. It will be more useful to me. I will look into your suggestions.

Hello Srichandrasekar

I agree with Arman. What is the protein you are interested in ? What is the size of your protein of interest ? Protein properties affect its ability to bind to the surface of the membrane. Factors like the rate at which the membrane will bind to the target protein and the capacity of the membrane to retain the protein need to be considered. Therefore optimization and experience counts as Arman rightly mentioned.

Nitrocellulose membrane are available in pore sizes of 0.1um, 0.2um or 0,45um. For proteins of mol.wt. less than 15 kD you can use 0.1um or 0.2um pore size membrane.

Thank you.

Smaller proteins will transfer quicker than larger proteins and possibly shoot through the membrane if you are running the electroblot too long. If you're afraid of losing your protein, optimize the timing of your transfer (V x hrs) and possibly stack membranes (two of the same pore size or the bigger holes towards the gel).

Another option is currentless transfer where you have one membrane at each side of the gel and use just filter paper to create a flow of buffer out from the gel.

Instead of NC, you should use PVDF. Higher protein binding capacity and affinity combined with better chemical and mechanical stability. 0.45 um is usually appropriate.