Dear Pranothi,

I have performed comparable protein extractions from various rat tissues, and - depending on the protein I tried to detect - I got good results using a RIPA buffer with a standard formulation (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Triton X-100, supplemented with cOmplete™ protease inhibitor cocktail). Could you please give us some additional specifications (e.g., buffer composition, loaded protein amounts, western blot protocol), that we get a better insight into the potential issues you may experience?

Dear Jonasz Jeremiasz Weber

I used commercial RIPA buffer from thermo scientific supplemented with protease inhibitor. Loaded 25ug and 50ug of protein for a comparison. I used semi dry blotting at 25V for 30 min and incubated with primary antibody overnight.

Dear Pranothi Mulinti,

Your approach looks sound so far. But I have a few more questions:

Have you performed a control staining of your membrane after transfer using a protein-dye such as Ponceau S? Moreover, what kind of gel type and running buffer did you use for the electrophoresis? And did you experience any problems when loading your samples on the gel, e.g., a jellylike appearance? (I suppose you use protein homogenates for detecting transmembrane proteins such as occludin.)

Looking back at my experiences and comparing them with your detection, I would primarily guess that your proteins might be overloaded. However, 25 µg of the sample does not sound too much.

Yes, I've cross checked the western blotting conditions and that seems to be fine. so finally I decided that the sample prep might not be good. Ponceau stain also showed almost the same pattern as shown in the blot picture here.

I took almost half the tissue (typically it says 5mg but I guess I took way more than that) in 400ul of buffer and homogenized using bullet blender.

I used 4-20% gel with MOPS running buffer at 200V for 40 min. I think 1 or 2 samples were little jelly like

As your Ponceau S staining showed a pattern comparable to your antibody detection, you can at least exclude an antibody- or target protein-specific problem. The SDS-PAGE conditions also sound OK to me.

However, the jelly-like appearance of your samples might constitute a problem for a smooth run and proper electrophoretic separation, leading to the observed smear. Have you tried to disperse the samples after heat-denaturing by direct, short ultrasonication or a comparable method? That is a standard approach in our lab when running protein homogenates on an SDS-PAGE. You may also try to start the gel run at a lower voltage (e.g., 60-80 V) until your proteins are in the gel and then increase the voltage again.

I have one last question regarding your protocol: How did you measure the protein concentration of your samples?

I haven't tried to disperse the samples. I'll try these suggestions and see how it goes.

I used BCA assay kit to measure the concentration