hello everyone
i have a high background problem in SDS PAGE.
i used 2-mercaptoethanol and SDS to denature the IgG, BSA sample (in 1x PBS)
and i boiled it for 5 min
i run the gel 100 V for 10min followed by 150V for 40min..
i don't know why it doesn't work.
does anyone know why it doesn't work?
thank you for reading
We will need some more information to help you with this.
- Is this a homemade gel or did you buy it precast?
- What percentage of polyacrylamide?
- Which lanes were samples loaded into?
- Was a ladder used? If so, what ladder and how much?
- How much BSA did you load?
- Did you use a loading buffer?
- What was the gel stained with and for how long?
- Was the gel destained?
This gel looks like it was stained with Coomassie Blue. It's normal for them to look blue or purple after staining, so this might not be excessive background. I can't see anything on the gel that looks like a protein band, and I don't know what the clear band towards the bottom of your gel is. Could it be tape? A common mistake is to forget to remove the tape barrier from the bottom of a pre-cast gel cassette, causing the gel not to run at all. Without a ladder, I can't tell if this gel has run at all.
Sorry for so many questions - happy to help with troubleshooting, just not quite enough here to give you an answer just yet :)
I noticed that you gave information about running the gel, but no information about staining the gel after it was run. These parts of your procedure should probably be subsequent, and independent steps. One consideration, is that staining tends to involve de-staining and you did not describe this part of your protocol either. Tube gels may be easier than slab gels. After running, staining
and de-staining the tube gels can be photographed side by side.
Hmm, where is your ladder or protein marker? I don't even see that present (often you run a pre-stained protein marker so you can see it while the gel runs). Did the ladder show up when your gel was running? That would be one place to start. Also, did you stain your gel after running it? The gel is blue, was your loading buffer blue or where did the coloration come from? Not sure if what you are seeing is really background or not as you appear to have no bands at all.
Destaining is not proper, and you probably forgot to load a protein marker. Some bands are visible in the lower part after the broken line, but without marker, how will you know if these are your desired bands?
Quick question, did your gel have a stacking gel layer and a resolving gel layer? Yours looks like all the same gel (often you see a color/stain difference between the two). Did you buy the gel or make it yourself?
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