Hi guys I'm out of ideas here please help!
I've never had consistent problem like this doing westerns before and I can't figure it out.
The condition used:
- First 4 lanes have 50ug protein in 20uL loading dye, last 4 lanes have 30ug protein in 12uL loading dye.
- 10% gel run at 175 V for ~1hr
- samples are total cell lysates from mammalian cell line in RIPA buffer
- samples are acetone precipitated. Loading dyes are added and boiled at 95C for ~6 min and made sure no pellet left
- semi-dry transfer at 0.2A for 1hr
- shown picture is blotted against actin. This antibody is normally really good
I re-made all my buffers just to be sure but no change. What else am I missing here?
Thanks!
Hi Cynthia,
That's a shame. But usually when I get "W-shaped" bands, I usually go back to my run. So, I'd check/adjust the following:
1) the wells are all nice and flat - I gently wash my wells with some running buffer to get rid of any residual acrylamide.
2) I run my gels at a lower voltage of 100V for a longer duration (1h30-2h).
3) I do a wet transfer at 100V for 1h30-2h.
The bands should hopefully come out nice and crisp.
This is unlikely to be the problem but:
I also dont do an acetone ppt. Instead I sonicate to solubilize my proteins by sonication, and remover debris by centrifugation - but that might be because I am mostly interested in chromatin-bound nuclear proteins.
Hello Hsin-Wei Tseng
You may have to do the following:
1. Make sure that your samples do not contain any traces of acetone.
2. Check the pH of the loading dye and the running buffer. Difference in pH can cause such distorted bands.
3. Reduce the voltage. You can use 70V for the stacking gel and 120V for the separating gel. Make sure that the power pack is applying constant power.
4. It need be try to load a maximum of 30 ug of protein per well. I feel 50ug is too high.
5. Try doing a wet transfer as mentioned above.
Regards.
Hi Hsin-Wei Tseng
Apart from Western blot, run a gel for SDS page.
If ladder and samples have both of these conditions, you should reduce the voltage or check the pH and concentration of the buffer running compounds.
If only the samples have these conditions, you should check the pH and concentration of the loading dyes compounds.
If there is no such problem in SDS page and you only see W-shaped bands in Western blot, you should check the transfer voltage, pH of the transfer buffer and its concentration of compounds.
I hope I was able to help you.
Good luck.
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