I am working on SDS-PAGE for proteins and observed 'smiling bands'. Is this happen due to temperature generated during electrophoresis?
It is usually due to excessive protein load in the lane. Try load less amount (and less volume) in the proper wells made with combs separated enough. It will also help run the gel "slowly" (not at high voltages) at the beginning until the bands reach the separating gel. Hope it helps
It is usually due to excessive protein load in the lane. Try load less amount (and less volume) in the proper wells made with combs separated enough. It will also help run the gel "slowly" (not at high voltages) at the beginning until the bands reach the separating gel. Hope it helps
I find that bis-tris gels are more forgiving and generally give prettier results over a wider range of sample quantities and qualities: http://openwetware.org/wiki/Sauer:bis-Tris_SDS-PAGE%2C_the_very_best
Sometime when you perform SDS-PAGE setting of v and mA is important and due to some problem it may changed. check the V if they are going higher then set it on 15 mA 30 V and run the gel.
Use about 35-40 micrograms of protein in each well and be careful at the time of isolation of protein, probably it may be due to degradation of protein, thus use the protease inhibitors in extraction buffer.
Protein degradation has nothing to do wid smily effect...alwez use ice packs to keep temp low around gel plates...also do not cross voltage above 80...give enuff time for polymerization...it shud take atleast 15-20 min to polymerize a 10-12% gel
The temperature generated during electrophoresis could produce an "similing" electrophoresis front instead of smiling bands. I seems that the problem could be due to protein concentration (probably yoo high) or excesive salt concentration in the buffer where you have your protein disolved. Avoid also to leave empty wells between yor sample wells. Fill empty wells with the same volume of 1X Laemmli's buffer that your using for your samples.
Usually 'smiling bands' occur because of two main reasons:
1. The PA-gel casted (both stacking and running)
2. the proper pH and salt constituency of the running buffer.
please chk and ensure both of the above facts.
... and propper cooling (at 15 °C but not below: SDS will crystallize at lower temperatures)
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.
Do electrophiresis at very low voltage, if separating gel is 12 percentage or below, I will suggest you for 50 , while if gel percentage is higher you can use up to 80 voltage
Always try to load sample diluted form with the protein extraction buffer. May it helps to get smile free band.
Use about 35-40 micrograms of protein - that's a lot. limit to 5-10 ug to get a clear bands.
1. Quality of separating gel
2. Load : 5 to 15 ug to get proper bands, overloading will also impact on band curving.
3. Mixing of load sample from one well to 2nd well also leads to this type of effects.
4. Running conditions (volt) : use controlled volt (not high) till bands reaching to separating gel from stacking gel. (High volt may lead temperature rise which effect on band shape and separating gel quality).
5. Right reagents , solutions and buffers use.
Hope this helps.
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