I prepared 15% resolving and 4.5% stacking gels on the day before running SDS-PAGE and stored at 4°C with wet-paper towels and wrapped with cling wrap. Noticed the band broadening effect while running my SDS PAGE, with an unusual line in the running gel (which is not clear in the picture) that progressively moved with running resulted in unsmooth pattern of bands. What precautious steps could I take to avoid these observations to get a better resolution with clear bands?
Hi Navaneethaiyer,
Do you perhaps have a picture of your SDS-PAGE after you ran in when it is not inside the tank? From the picture you posted I cant really tell if there could be a problem with the way the running/stacking gel set. I had a similar problem with distorted bands when running SDS-PAGE gels that was caused by too much voltage when running the gel. The general rule of thumb is to use 10-15V for each cm (length) of your gel, but if the bands or lanes warp, reduce voltage and retry.
Band distortion may be resulted from temperature rise. You can try to run your gel in 4C refrigerator. One of my collegues always runs his gels in refrigerator (if there is a plug inside)
I want to add one more point to above answers , try running gel with less concentration of protein in sample.
Hi
what buffer are your samples before you add sample buffer? components from buffers (salts, surfactants, etc) often cause band spreading and lane encroachment. sample overloading can also cause these symptoms.
Hope this helps little
Thank you all.
Dr. Riegardt Johnson! Kindly find the snap of the gel (after having the proteins transferred to the PVDF membrane for WB) and the molecular markers on PVDF membranes. I am sorry, but I did not stain at least one gel, and used both in membrane-transfer. I ran SDS PAGE with constant current of 20 mA/ gel, with 200 V and I am sure that there was no significant heat produced. Membrane shows clearly that the markers have broadened and these observations were noticed during my second attempt too (similar conditions with fresh 15% gels).
I am trying to evaluate the expression of my target protein following transfection and analyzed the cell culture media (for secreted protein) and the cell lysate (for intracellular expression). Post-transfer gels show that the samples from media have some unusual wavy bands (corresponding to BSA from FBS), while the gel area for lysates remains clean.
Dr Oliver W Bayfield! Thank you for your advice. Do you have any idea for these observations?
Thank you.
Do not pH your buffers. Check the pH and if it is way off, dicard and redo it adding the correct ingredients. Slowing down the volts as suggested helps too
Try to change the stock of acrylamide, and bis acrylamide monthly, it forms aacrylic acid if not.
Make running gel day or so earlier, overlay with water saturated N-butanol to get sharp demarcation line, wash away N-butanol with running gel buffer and keep it at RT with upper entrance well sealed to prevent evaporation. Make stacker before use. You should not keep gels with SDS in the fridge, especially when the temperature is well bellow 8 C, since SDS changes at low temperature. RT is OK for gel, but never make the whole gel to keep it longer than 2h or so. Use it! Otherwise, You will have buffer diffusion between the stacker and running gel and will lose the major Laemelli effects - stacking and resolving induced by differences in pH and concentration of buffers.
Another consideration is contamination of the sample with DNA. If a lot of DNA is present in the sample it can cause physical distortion of the banding pattern due to interference. To reduce large strands of DNA present, simply treat with benzonase.
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