Our lab recently switched from semi-dry to wet transfer system for western blotting. For some reason, our lower molecular weight bands always appear as frowns or unhappy face shapes (around the size of beta-actin). i.e. they are not straight. Does anyone know the potential reason for this? We tried reducing the voltage during running but this did not help. Any ideas? We are using the bio-rad mini trans-blot system and perform our transfers in the cold room at 400mA for 1 hour. Thanks.
Hi Andrew,
The smile effect (happy or unhappy patterns) of the band happens when the migration of the proteins during electrophoresis become either (1) too fast or (2) too hot. Both factors might play as well contributing towards smiling pattern. The proteins get transferred to the NCP/PVDF with a same signature mark the way it had been electrophoresed in the gel.
You can reduce the migration by reducing the current ( I use 20mA/plate for Bio-Rad Protean II plates) and you can also check for the desired pH of the resolving gel and the running buffer. Heating while the gel runs can be minimized either by running in cold room or by keeping the electrophoresis apparatus covered with crushed ice.
I would run 2 gels for western blot, 1 for Coomassie blue stain, the other for western transfer. This tells you if your smiling is during SDS-PAGE or not. It is also generally useful for seeing how well loaded the gel is. I always chill my electrophoresis bufffer and fill the outside chamber as near to the top of the gel as possible to keep it cool while it runs. If you mix your own gels, try using fresh TEMED and APS.
Hi Andrew, Amritial, Paul, and Danuta have given great advice and I would flow their suggestion. I also want to support Pete's comment about fresh APS. If making your own gels, having fresh/made the day of APS is really important to the quality of the gel. For the rest of the components stocks can be made, but APS should always be made fresh. Best of Luck!
Hi Matthew,
I agree with Pete that using fresh APS is definitely a good idea and I also prepare fresh 10% APS solution before casting the gel and keep it protected from light for its photosensitivity. But I don't know what did he mean about fresh TEMED. TEMED comes in liquid form (Sigma Aldrich T9281-25ML) and I just directly add it from the bottle to desired concentration.
If TEMED is kept around for a long time, it can go off and the gel will not polymerise properly. A few times I've had gels remain liquid and this has resolved with using new TEMED or someone else's stock. It may not happen often, it may have just been down working in a lab and using up previous researchers' leftovers.
If you have space load the markers on both end side wells of your samples. This can reduce the smile/frawn face of the samples. If you add same amount of loading buffer on both end side wells of markers you can reduce the smile/frawn face of your samples and markers.
Hi there, in my opinion the pH, voltage and heat issues should be resolved for smiley effect of bands. I think we need to know that your individual band are of sad effect or the gel on the whole? i.e. band on left and right sides of the gel are moving fast and the middle ones are slower.
Regarding using fresh APS will not do anything with these bands, as I prepare 10% APS and store aliquots (150ul, required for one running gel 10% and one stacking gel 5%, ) at -20 for a 8x10 cm gel. See the attached Ponceau-S stained protein transferred on PVDF membrane.
I would suggest to wash wells too after polymerization of stacking gel either with distilled water or with 1x Tris-Glycine-SDS buffer. This will remove any unploymerized gel solution from the wells, which can hinder in good electrophoresis.
Good Luck
I agree with Zulfikar : does the whole pattern smile across the gel width, or do individual bands appear crooked ? In the first case, reduce the migration voltage to prevent overheating. In the second case, the problem is more likely due to the composition of your sample : too much salt, wrong pH, too much protein ? At any rate, it sounds like the problem arises during migration, since it is hard to imagine how it could occur during transfer. Regarding fresh APS solution, no need to be too fussy; the solution keeps for a few days at 4 °C. You will be able to tell if it has gone bad if your gel takes too long to polymerize (>20-30 min). Beware that the powder itself is hygroscopic and will decompose in the presence of humidity, so buy small batches and discard old batches if they are caked and need a hammer drill to scratch a few crystals.
I will suggest not to use the SDS gel immediately after preparation. Let it to stabilize its mesh network while keeping it for an hour at 4 °C. Also use the freshly prepared SDS running buffer for atmost 4 times. Run the gel on 90 V for 90 min and then raise to 100-110 for 40-50 min. Dont reach the gel running to the glass bottom. I hope so, it will solve your problem.
Nice to read so many suggestions.
I would like to add one point that may be helpful.
During first 15-20 minutes of run, when the samples are passing through staking gel keep the run at 90 volts. After that you can increase the voltage to 130.
I use protein extraction buffer with loading dye (loading buffer) at both ends to get rid of the smiley bands. Sometime add this loading buffer in the ladder when lanes are limited. Hope this helps for others too.
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