Western Blotting from Protein Extracted from Organs Tissue with High Lipid Content

[rizky]

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Thank you for your response.

I used the tissue from experimental model. I think I couldnt use from cell line.

lipid is also my consideration. from Western blotting result, what kind of description that might be resulted from lipid interference?

Thank you

[rizky]

Previously my senior used the primary antibody to another samples, and it worked with no or less background. I followed protocol precisely
-blocking in 5% milk 30 min
-1st Ab 1:500 dilution in TBST overnight
-2nd Ab 1:5000 dilution in 5%milk 1 hour
3x wash each 10 min after 1st and 2nd ab

there are several things that to be my concern
-the milk powder was not mixed well; although I used stirrer for more than 1 hour
-antibody aggregration; although I always pippeting before apply to the membrane
-residual milk after blocking

thank you for your suggestion
best regards
Rizky

[admin]

Your transfer is very dirty. I would use clean or new casettes, buffer, and sponges. Also running seems to be an issue, possibly use new fresh running buffer and careful prepare it to the right pH and buffer recipe.

What paper/membrane are you using for the blotting? ie from what company and type - nitrocellulose vs PVDF?
you may need to prepare it before transfer and carefully block after.