Hi to all, I had a problem with my resolving gel (SDS-PAGE) with my last two gels.
I preparedtwo time all the solution and buffer, I followed the maniatis recipe, preparing 8% gel resolving gel and 5% stacking gel, I didn't have problem with the polymerization (new APS), or during the run (45V constant stacking, 100-110 resolving). After the transfer (about 1h on ice at 60Vcostant, setted protocol). I controlled the gel with coomassie, the nitrocellulose with ponceau and I had a sad surprise (Fig 1-2). I compared this figure with the coomassie (after transfer) and ponceau of an old gel (same protocol) of last year (Fig2-3). The samples were different (muscle biopsies, 40ug) but with same lysis (SDS+Urea) and same laemmli recipe. The samples were boiled at 95°C and vortexated, I never have problem with this preparation that I used for dysferlyn and calpain analysis, same for marker, disappeared (as in this case) or bands were not sharp and distinct.
I didn’t see the correct protein pattern, I didn’t have bands sharp and distinct at high molecula weight. I analysed a protein at 43 KDa and I got a good signal so I didn’t think about protein degradation. I had also a strange coomassie (only after transfer) behaviour (fig3) there were a sort of smear around the myosin
Unfortunately I had to use very old SDS powder and old expired acrylamide solution (always used Sigma solution, correct concentration), could it be the reason? I will check again the Tris pH, even if it is fresh prepared.
I think about try to run two gel one with sameTris buffer solution but find different SDS or acrylamide, analysed with coomassie. I didn’t understand if could be a pH problem or one of the old reagents.
Help me with your experience please!
Thnaks
Valeria
Yes Valeria, the SDS is always critical, prepare a fresh solution with new reagent and store it at 4C, In the first gel your proteins seems to be un-denaturized, in the last gel your gel looks good. In you case your protein has high molecular weight so you need good sds, and much more time to transfer. Best, A.
Hi, thanks for the answer and the suggestion!
In this particular gel I analysed protein under 50KDa, so I prepared a transfer without SDS and only 1 h for transfer, but in the second "terrible" gel (not show) I have to analysed high molecular weight protein so transfer with SDS and more time and voltage. In both case of transfer with and wihout SDS I had same result (band not sharp and well defined after coomassie and ponceau), regardeless of time transfer)so I am fixated that my problems egards gel or running step instead of transfer step (maybe erroneously !). Your observation about un-denaturized, even if I had boiled the sample, give me a great hint to verify other aspect. I prepared laemmli handmade fresh aliquote w/o SDS, I stored them with beta mercapto at -20°C. it is better to prepared new one for my test!
Dear Valeria,
In addition to the excellent Adrian’s suggestion, you should use fresh solutions always; they are ok for several months anyway. The Laemmli buffer is better to store at -20°C without beta mercapto, and add it before to use (then you can store it at 4°C).
Good luck. Cristián
Thanks Cristian, I will follow your suggestion. In general how long you store your laemmli aliquote with beta mercapto at 4°C?
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