I use western blot of EGFR and p-EGFR using skin tissue.
By the way, the band does not come out clearly and the background is too dirty.
We also used 6% SDS-PAGE gels and used 4% -15% grandient gels. I am transferring from 40V to 2 hours, but recently I am trying from 60V to 2 hours.
The membrane is nitrocellulose, and the transfer buffer is tris, grycine, and methanol. (I did 20% of methanol and tried 5%.)
I have tried blocking 5%, 3%, 1% skim milk or 10%, 5%, 3% BSA but it does not work well.
How can EGFR and p-EGFR western blot in skin tissue be able to get the band out?
Thank you for your advice. Thank you.
Hi Jinju,
Better to look at each step before drawing any conclusions!
1. Sample preparation: Make sure that the lysis buffer is good enough to lyse the cells (or else use the sonication). Add 10% of Mercaptoethanol and boil the sample at 93'c for 5 mins. Quantify your protein before you load it into the gel.
2. Add fresh APS to prepare both stacking and resolving gel.
3. Keep the transfer tank set up in a container with ice; so that the temperature will not affect the transfering of the protein from gel to blot.
4. Use 5% of fresh milk solution (prepared by using TBST) to block.
5. Prepare your primary antibody with the same 5% milk solution for better result.
6. After overnight incubation of primary antibody at 4'c, wash thoroughly with TBST (3 washes for 10mins each).
7. Check for suitable secondary antibody dilution.
8. Wash your blots thoroughly with TBST.
9. Make sure that you add ECL just before keep for imaging!!
Hope these tips would help you get better blots.
--Best regards.
Hi Jinju,
Could you provide us with the brand and catalog number of your primary and secondary antibody as well as, if possible, an image of your defective blots. It may help to spot possible problems.
Best,
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