Hello everybody,
I have been using Hoefer SDS PAGE system. It gives fantastic bands. But the only problem with this system is that it's combs are open which causes it's wells to shrink down. It decreases the well capacity and also seepage of sample into other wells. Can you please suggest me, how can I fix this problem.
REGARDS
MAZHAR
Hi Mazhar, most gel casting systems are designed in this way and you will always have some loss of polymerization on the top part because oxygen from the air quenches the polymerizing radicals. We normally try to fill up the stacking gel to the limit of the glass plates, so that when you insert the comb, very gently, you have an overflow and you will form a tension bubble of stacking solution on the top of the glass plates. If you keep the gel in a cold room you'll also reduce evaporation of the water without affecting the polymerization that is actually favoured being an exothermic reaction. You will still have a bit of well size reduction, but should be minimal. We are normally able to load up to 30 ul without spillover risk.
I hope I made myself clear and it helps! Cheers and good luck!
Hi there,
The fact is that your comb has much longer teeth that needed therefore the gel your are preparing is in contact with air during polymerization. One solution is that your comb should fit exactly onto the white plate so that there is no contact between gel and air (the Biorad device is perfect from this point of view, watch the video : https://www.youtube.com/watch?v=EDi_n_0NiF4) so you may design and cut a new comb which allows to form the expected well capacity and disallowing contact with air.
As mentioned above, its not unusual to have incomplete polymerization at the top of the stacking gel. You could try degassing your stack mix just before you add the catalysts. But, if you do this you actually risk the stack mix polymerizing before you have finished transferring it and inserting the comb. You could also try adding a bit more of the TEMED and AP. Another solution, which actually depends on the protein concentration of your samples, is to just load smaller volumes.
Hi there,
I agree with Bruce. Add a bit more of the TEMED and AP. You can increase for 1.5x for each one, but take care. Your gel will go polymerized more fast (5-10 min.). So, make the mix and aplly immediately.
Hi there,
Many great answers already! I do the same as mentioned before: apply a bit more of TEMED (work fast, have everything ready) and add the stacking solution until the edge of the glass, when you introduce the comb it will overflow but you will make sure the wells do not shrink too much. Another tip could be to place the left over of the stacking in a vortex very gently so it takes longer to polimerize and every 2-3 minutes you can apply more solution on the top of the gels. With this, you make sure the wells are deep enough, but it will take longer time.
Hope it helps!
best,
Hi , I also used to face the same problem before. The easy way to avoid shrinking of wells is, make little more stacking gel than needed. After inserting the comb, pour some gel over with the help of 100ul pipette.
As I can see from the pic your comb is staking on the base but still there is some gap in between. To reduce this you can just cut the comb from both sides and let it go upto just above the plate/gel. by this way there will be minimal contact b/w gel and air.
Also as Bruce and Murilo mentioned, you can increase the TEMED and APS but that can harden your gel.
Hope it will work..Cheers !!
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