I have performed venom fractionation using HPLC and run the samples in SDS PAGE. I wanted to know what could be the possible reason for my protein bands to look like this. It should have been crisp bands.
There are quite a few possibilities:
1. Your samples might have some chaotropic salt such as urea or other such elements.
2. The pH of the gel or running buffer is slightly acidic. Make sure the pH of the running buffer is 8.3 or higher. Usually, titration to 8.3 might make the gel band fuzzier. I have noticed that the buffers work the best around 8.8 despite traditional methods suggest adjusting it to 8.3.
3. Was the gel solidified properly. You can try cooling the buffer and the gel before running and if running in a wet apparatus, try keeping an ice pack inside the tank or simply run the apparatus in cold room. Heating/melting of gel might also lead to fuzzier bands.
4. This is related to the above point but may be the gel was running at a higher voltage and therefore melted. Try running at lower voltage in cold room.
5. This might sound naive but can actually be true. Have you checked your blot with coomasie before staining? And were the bands crisp then? If yes, it could be an error with the gel doc! Sometimes the gel doc camera has fog and disturbs the focus.
No urea was added. Gel was solidify properly. It was run at 80v. I think its because of the SDS loading dye. Doing it again with freshly prepared dye :)
Hi Siddhath,
Where from did you isolate your protein (brain, liver, a cultured cell line ...)?
What is your gel concentration?
What conditions at which you run the samples (constant or variable volt , current, time...)?
Looks like something to do with the components present in the reconstitution solution of HPLC purified venom components, since the other 3 lanes looks to have ran fine. I assume you have been doing this numerous times by now, do you think acetonitrile/TFA if not removed completely can cause this? May be do a buffer exchange of the sample if you can still spare some.
Thanks everyone for replying. So I figured out what the issue was with this gel. The sample loading buffer I used contained less glycerol, so the protein wasn't properly stacked. I made the buffer again and it worked fine. Mohamed Khashan These are RP-HPLC fractions (TFA and ACN-running buffers) of venom proteins and I ran at constant voltage.
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