What's the problem with this SDS-PAGE? - (Jul/29/2009 )
This is an image of 12 % SDS-PAGE of crude bacterial (E. coli) lysate. The protocol from ‘Molecular cloning: a laboratory manual’ by Sambrook & Russell was followed for carrying out PAGE. The gel dimensions were ca 17cm x 17cm. 40ul sample mixed with 20ul of 2X loading dye and denatured for 5 minutes at 95C was loaded. We would have actually used 40ul of 2X dye, but previously 20ul has worked very well. We did not estimate the protein by Bradford's method or so. Can anybody comment on why the gel has run so badly?
ram on Jul 29 2009, 02:34 AM said:
Assuming there is nothing wrong with the gel itself, I think you ran too much lysate. Occasionally I get similar looking gels when I overload them. Typically, when I run crude lysates I run a lot less. I don't quantitate either, but my method is as follows: pellet 1 ml of bacterial culture, resuspend pellet in 25 ul PBS, add 25 ul 2x SDS Dye and boil. From this, I load 5 ul. Sometimes, I have to play with this a bit (2.5 ul - 10 ul) since the bacteria are at different densities when I harvest.
Attached is an example gel using my method (5 ul each of crude).
I would definitely say overloaded.
Did you run a size standard, and if so, did it look good?
Yeah, too much loaded and probably too much denaturing agent in your loading dye too.
Thank you all for comments.
Roo, yours is a really nice picture...
Can overloading be the only reason? In the last lane of the gel, there is a low-concentration sample loaded, that too hasn't ran very well! Is it correct if I say that at least lower bands (which show relatively lower intensity) should have been clear (although little hazy) even if it is a case of high loading?
No I didn't load any standard marker...But I had put BSA in the middle (5th) lane since it has same size as that of protein of my interest in the bacterial lysate, and u can clearly see what has happened to this lane!
ram on Jul 30 2009, 09:05 AM said:
Is this a precast gel or one that you made? I tried doing the 2nd dimension of a 2D-PAGE on a precast gel that was past the expiration date, and it looked horrible.
Although it looks like the gel is overloaded, I have also seen this when I accidentally miscalculated and loaded too much loading dye (too much SDS). Occasionally, you see this if you run your gel too fast/high or if your gel is bad. If you are using commercial pre-cast gels, then just try a different batch. If you are casting your own gels, then try remaking all of your reagents just to make sure one them hasn't gone bad on you. You might even titrating the loading of just one sample to find your optimal loading range. Sometimes it will take you a couple of trial and error gels to find out what works best in your hands.
Its not a pre-cast the gel, but it has been made in the lab according to the composition given in ‘Molecular cloning: a laboratory manual’. I forgot to mention that previously we have got quite satisfactory picture as attached below, with the gel made from the same batch of acrylamide mixture prepared in the lab. Yes, we ran the gel at little higher (?) current: 35mA