I am trying to analyse proteins qualitatively using sds page laemmli method. Where in this particular case I am using 15% separating gel and 5% resolving gel. The sample loading buffer is as 0.055 M Tris-HCl, pH 6.8, 2 % (wt/vol) SDS, 20 % (vol/vol) glycerol, 4.3 % (vol/vol) β-mercaptoethanol, 0.0025 % (wt/vol) bromophenol blue G-250. On constant voltage setting of 80 volts till dye crosses separating gel (approx 50 min) and then 140 volts till it reaches bottom (approx 2.20 hrs). Due to smile effect and improper resolution of lower marker proteins of ladder, and also broad bands on my unknown sample on my last couple of runs at 12%, I decided to increase gel percentage to 15% and lower the temprature by keeping reservoir tank in an insulated box by adding plenty of ice+water+salt (salt to avoid melting of ice) to submerge 80% of box so as that electrical contacts and top 2 cm of reservoir tank does not comes in contact of the water or ice.
My thoughts regarding this bizarre effects are;
1. If we look up closely on the top of the image their I can find some portion of gel itself wavy, leading to improper differentiating line between separating and loading gel.
2. The temp for run was too low. may be around 0-4C since ice barely melted (not more than 20%). As SDS starts precipitating at lower temperatures below 4C.
3. The vertical dragging of protein marker in extreme last lanes may be due to the air present during polymerisation of separating gel which lead in filling of some of stacking gel therefore lead to it.
4. Do I need to lower the gel% or protein concentration?
Pls note: Ignore the background stain, I stopped destaining as soon as these bizzare bands were visible.
Looking for some explanations along with your suggestions and comments.
Hi
two suggestions
1- the polymerization of gel maybe not completed
2- use 200 V
you can get similar effects either with too high a current or by loading the samples too slowly and waiting too long before starting the run allowing the smaller proteins and salts to diffuse sideways into the gel.
Too much salt in the samples will also cause poor banding
Thank you jasim I do think incomplete polymerization might be the issue. In terms of such high volateg I am bit skeptical never tried with that much.
Paul the voltage (140) was comparatively low since it was being run a temp between 0-4 so lateral or vertical diffusion due to resistive heating can be eliminated. I am not using any salt for precipitation.
I did use 20%glycerol though.
I agree Kartikey Chaturvedi but the voltage is secondary in that the current is what causes local heating so on the voltage issue I was querying really if the correct buffer/concentration of buffer was used in the gel. I do not think that glycerol will make any difference
Paul Rutland Thank you for your reply. In a previous run I tried for 50ma for my stacking gel on the unit what was showing 19ma for the actual.
On response to the buffer concentration here under was my recipe
For sample gel 5% volume of the components (μl) Milipore water 5700, bis-acrylamide solution 1700 Buffer 2500 (0.5 M Tris-HCl buffer of pH 6.8), 10% (w/v) SDS 100, APS 50, TEMED 5
For separating gel 15% volume of the components (μl) Milipore water 1400, Acrylamide/bis-acrylamide solution 5000, Buffer 2500 (1.5 M Tris-HCl buffer of pH 8.8), 10% (w/v) SDS 100, APS 50, TEMED 10
running buffer 1x trisbase 3.03g, 14.4g glycine, 1g sds in 1ltr millipore water.
After pouring separating gel left it for 20 minutes for polymerisation and after pouring stacking gel left for 30 minutes for polymerisation.
Since, I was running it for two gels in single buffer tank the time was less than 10 minutes. run started as soon loading ended.
Paul Rutland Is it possible for you to see the image up close specifically in the top area, are there signs of incomplete polymerisation ?
or is it possible that the since the reservoir tank was kept at below 4C the sds precipitated and lead to such run?
Thank you for the detailed information Kartikey Chaturvedi . The Buffers look fine
The wells do look too rounded and not straight edged Comb shaped at the bottom of the well so I think that you are right about polymerisation. Hopefully you use fresh APS evry couple of days and if so perhaps waiting a bit longer for full polymerisation might help as Jasim Al-saadi suggests. One other thing is that PAG gels leak non polymerised acrylamide into the wells so it is very important to wash this out of the loading positions with buffer so that the sample loads on top of the gel and not on top of the excluded liquid and there is a clean electrical contact between sample and gel.
Salt iced water does get very cold ( down to -20c) but at 0.1% I do not think that it will precipitate but then I have never tried that method of cooling. My guess is that the local heating effect of the current in the gel would stop this happening
Dear Kartikey,
Prof. Paul's advises are accurate. I happened to run through a similar problem during early stages of my research. Later, I happened to realized that these could have been the issue with the gel: Improper polymerization; frozen gels; no SDS in the running gel buffer; inappropriate power source causing over heating of the gels. The link below may help you more.
https://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/bizarre.html#2
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