Looking at your gel picture, I would suggest you to do a few things:

1) Use a high speed, temperature controlled (4 degrees) centrifuge while extracting your protein

2) Maintain the low temperature throughout

3) Try centrifuging your protein again and carefully take the supernatant, just before loading on to a gel

4) Use a 12% resolving gel or a 4-20% gradient gel for SDS-PAGE

5) Make sure that you reduce your protein before gel loading (95 degrees for 10 minutes in loading buffer)

Good Luck!

I would say these smears are nothing to do with protein amount (I used even 10ug) or volume of loaded lysate. More likely problems are with the gel, running buffer, lysis buffer or sample buffer. Did you try, by the way, to stain the membrane with Ponceau? Were all your bands there clear and sharp?

What I would do in such situation is to prepare all fresh reagents or ask colleagues' working solutions. Also one could think to change these tubulin Abs. There are other housekeeping genes, which can be used for a loading control. Good luck!

I agree with the above comments. I only want to expand on Rohit's point 5 and stress that you make sure you have both enough SDS in your loading buffer (final concentration greater than or equal to 1%, though I routinely use 2%), and sufficient reducing agents (5% v/v beta-mercaptoethanol or freshly added 100 mM DTT).

A couple of other special points to bring up just in case:

1) I've also had problems in the past if I was dealing with lysates that had a lot of lipid present (fatty tissues or adipocyte cell lines) and that can cause smeariness when RIPA was used as this will dissolve more lipid than milder lysis buffers.

2) Is your boiled sample viscous as well (goopy instead of fluid) and difficult to pipette? That can also lead to smeariness because the RIPA denatured the DNA causing aggregation. If your sample though is perfectly fluid then likely not an issue.