Hi Catalina Moreno . What is the source of your collagen IV positive control and antibodies?

Hi Catalina Moreno

I can't say that I've had a lot of experience in blotting for ECM proteins, but a quick search shows that not getting bands on your WB is a common problem. Luckily, it looks like there are a lot of good recommendations. Here are some links that hopefully point you in the right direction:

https://www.researchgate.net/post/Problems_with_Western_Blot_for_elastin_and_collagen_Protein_extraction

https://www.researchgate.net/post/Type-I-collagen-Western-Blot

https://www.abcam.com/protocols/collagen

Good luck!

Hello

Western Blot for ECM, collagen in particular could be tricky. A proper isolation method is important for a good Western Blot. You need to be extra careful with pH, temperature as well as concentration. Even selection of antibodies should be right. You need to re-visit your extraction protocol.

Best.

Do not trust to ladder only. Apply ponceau S staining and first see your protein of interest at membrane after transfer thereby primarily validate your extraction and transfer method. Afterward freshly prepare and adjust well the concentrations conveniently of your primary and secondary antibody buffers. Validate your loading concentration and imaging method by using a housekeeping protein if possible.

Emir