I tried to detect alpha tubulin, I used total protein (50 ug) from liver tissue with cirrhosis and/or hepatocarcinoma and liver tissue with cirrhosis and/or hepatocarcinoma associated to Hepatitis C virus infection. I stored the lysate on -20 degree and use a dilution 1:500.
Do you have any suggestion why the tubulin have bands of different thickness? You can see my WB result on my attachment.
There could be a number of different reasons. Protein may not be loaded equally giving different intensity bands, did you do a protein assay and check loading prior to transfer? If you run on a too low percentage gel you are likely to get smeary bands also if you run too fast. So you could try increasing percentage 10% should be ok or try reducing voltage and therefore speed of gel front. Something else to think of is whether your samples are properly denatured prior to loading. Someone who is better at Westerns than me will likely have some other explanations.
Check the following in orders:
Completely thaw the protein sample.
Determine protein concentration.
Add enough SDS/LDS and DTT, make sure to load the same amount of protein.
Use >= 12% gel, include a protein marker, let the alpha-tubulin be separated roughly in the middle of gel.
Use more sponges if using wet transfer, make sure contact between membrane and gel is tight.
Use enough solution of antibody to incubate the membrane when shaking.
Luisa, the most likely reasons are 1) Tissue is differentially homogenized, some being more completely homogenized than others (i.e., TUBA levels are reflective of degree of homogenization, 2) Tissue composition is different for each sample, some with more cells than others (i.e., TUBA levels is reflective of cellular content in tissue), and 3) TUBA expression is impacted by the disease conditions (i.e., TUBA levels reflect degree of pathology.
Other possible reasons are you have loaded an unequal amount of total protein, not likely assuming you have performed a good protein quantitation assay. If the protein levels vary widely between your samples, this could be a source of your variation, even with appropriate quantitation. Ideally, your lysates should have protein at a concentration of about 1-3ug/uL. Much less or more can result in some issues. Depending on what you are attempting to detect (very low abundant protein?), I don't know that 50ug of total protein is necessary. 10-20ug is sufficient for most tissues, and I go down as low as 5 ug for cells from culture. At or above 50ug you start to run into problems with protein migration, especially in mini-gels with 12+ wells.
To determine if you have loaded grossly different amounts of total protein then consider staining your gel with commassie blue, or your membrane with Ponceu S. These will show whether or not your loaded equal protein. I bet you have, so then you may consider the more likely reasons I've stated above for future tissue processing.
Best of luck.
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