Dear all,
I've been working on isolating membrane proteins - MPs from Leishmania donovani and I want to have a rough idea of how well resolved they are from one another, in principle, using a minigel system (12% polyacrilamide, tricine-SDS-PAGE by Schaegger et al, 2006). The method to isolate the MPs is briefly described as follows:
1) Lyse the cells (3x freeze thaw in liquid nitrogen plus five cycles of sonication - 15 seconds each with a minute interval between two cycles)
2) mechanically shear the DNA with a small gauge syringe
3) spin at low speed (1.000 xg, 4C for six minutes) to pellet down intact cells
3) collect the supernatant containing the lysed cells and spin them for at 100.000 xg for 1h05 (4C)
4) wash/ resuspend the pellets with PBS to solubilize any cytosolic protein that has been pelleted down
5) spin again at 100.000 xg for 1h05 (4C)
6) resuspend the pellets using a 2% octyl glucoside (in 10 mM Tris/ 150 mM NaCl, pH = 7.5)
7) spin for 25 minutes at 178.000 xg (4C)
8) collect the supernatant containing the solubilized MPs (about 4 ml)
9) top up the volume to about 20 ml with PBS to disrupt the micelles (octyl glucoside CMC = 20-25 mM at RT. A 2% octyl glucoside solution is about 68 mM, so that, after topping up the volume with PBS my concentration is about 13.5 mM i.e. below the CMC)
10) spin at about 1.300 xg using ultrafilter (Vivaspin 20 ml MWCO 3 kDa) to get rid of excess detergent (no longer associated in micelles) and salts.
Does anyone have any idea why my last band (image enclosed - MP L.donovani BPK282...MPs from L.donovani at the left hand side, next to biorad precision plus unstained protein standard) which is heavily glycosilated, becomes bowed? Is this quite normal when I am attempting to separate glycosilated proteins or is there any room from improvement on my method?
I also ran a longer gel and my last bands (not only the last) also became quite wavy. MPs are on the left hand side of the second file (MPIV1000C tall2...) Please note my heavily glycosilated bands (carbohydrate TallII_MP)
Thanks for the attention and best regards
Bruno
The first two images are protein stain (Sypro) and only the last image (carbohydrate TallII_MP) is a carbohydrate stain
Time for running the SDS PAGE is generally accountable for the bow like changes in the visualization of bands.
The reason is generally heating of the gel end here.
check the Ph of the buffer too.
Hi Shivani,
you are right, my next step would be to increase the voltage (I used 150V) to maybe about 250V to decrease running time (possibly running in a cold room to avoid overheating).
Nonetheless I still think there might be something related to my preparation itself. You see from the second and third picture that the lane on the right hand side, which contains the cytosolic proteins, also become wavy by the end of the run, yet way less wavy then the MPs.
Best regards
Bruno
Judging by the lack of the 'reverse smile' on the marker lane this effect is definitely something to do with your sample. The effect is only at the dye front suggesting that it is due to pH, salts and/or OG. If you put the protein marker into your final buffer you should see the same effect on the marker lane. You might be able avoid this effect by using gradient gels or different pH in your gel but to be honest if it doesn't effect your conclusions then it's nothing to worry about. The literature is full of gel pictures with various smiley artifacts! The longer run artifacts are most likely down to temperature increase and consequent uneven expansion of gel, edge effects etc.
From my limited experience on membrane proteins on SDS-PAGE I think you overloaded the gel. That band is "fat" with the central part negatively stained. Try to reduce loading significantly, or do a titration of protein concentration. When loading is right you will have a normally looking band. Of course, other bands of less abound proteins will be lighter as well. I hope this helps. Good luck!
Thank you both for the reply!
@ Deepah: unfortunately I do not have the option of running a gradient gel as the final gel that I will be running is a long one, not the mini ones out there. But as I explained previously, will run the next gel inside a cold room and increase the voltage to get a faster run
@Chao: It is indeed tricky because my bands' intensity vary a lot, as you noticed. As you can see, my band above 50 kDa (which is probablymy GP 63) is very strong whereas others are quite feint. I ran previously a gel with a lower loading and it ran fine until the last band, yet they were all quite feint.
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