I've tried to assess RyR2 in heart tissue by western blot but I'm not succeeding in such task, mainly because of its high molecular weight. The main problem is the mobility, despite the fact I used 4% (SDS-PAGE (stacking gel) and 6% SDS-PAGE (resolving gel), as widely described (run time: 6 h at 120 V + 6 h at 150 V) (see the attachment).
So, I need some assistance from people who had already faced a challenge like western blotting high molecular mass proteins.
Below I provide some details of my protocol:
· NP-40 and RIPA buffer were tested and no improvement could be seen;
· Concerning the samples, we homogenized rat heart in 50 mM Tris, 1% Triton, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF and protease inhibitor cocktail (Sigma S8820). After, samples were mixed with Laemmli buffer (final concentration of 62 mM Tris, 2% SDS, 10% glicerol, 0.002% bromophenol blue, 5% β-mercaptoethanol).
· A range of 20 to 80 ug was applied in each lane (6% SDS-PAGE);
· Up to 12 hours of running (120 V);
· 250 kDa marker entered until the half of gel;
· 500 kDa stayed stuck in the stacking gel;
· Wet electrotransference was run for 90 min 100 V and was not efficient. Some proteins were still stuck at gel, which was Commassie stained after the process.
Hi, you can use less % of resolver gel specially the Bio rad prepared gel 5% or less . Then run for 110 volt 90 min and transfer for 24 hours at 30 V .
How long did you extract your proteins? Was it carefully extracted on ice or at 4'C? From your total protein gel, the problem might come from the protein extraction step, as there are no obvious multiple bands.
Since your 500kDa protein marker stucked in the stacking gel while your 250kDa marker entered half of your gel, you might need to consider to use lower percentage of resolving gel. You can try with 4% and see how it goes. If you are doubt whether you successfully extract the protein, you can check the protein content beforehand or use silver stain instead of comassie blue to see the presence of your protein band.
I used to check purity of my peptide (smaller MW) sample and stained them using silver nitrate. If there were bands, I will replicate the procedure but stain the gel with comassie blue instead.
I agree with the comments given above. You need to use 4-8% percent gel for getting good resolution
Thanks for all replies. Below I address each point raised:
Md Main Uddin: we've tried a 4% gel for 5,5 h run at 120 V. HMW proteins just entered nearly 1 mm into the resolving gel (see the attachment).
Nurul Adhwa Rahman: the extraction was fast, on the ice, with protease inhibitors. There's no indication of protein degradation in the sample when running for other targets.
Muhammad Shafiq Ghazalli: we can see HMW proteins in the gel, with nearly 500 kDa. Commassie blue has enough sensitivity to stain this band (see the attachment).
As you can see in the attachment, a 4% gel allowed a small penetration of the HMW protein, that may be enough. However, I'm still having troubles to blot it in the PVDF membrane. I'll appreciate if you have further suggestions.
Michael
Folks
We've just performed a 2.5-10% SDS-PAGE (6 h run) and Ryr entered just 1 mm. We'll appreciate receiving tips from you!
Regards,
Michael
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