I ran a Western blot a few times for the GADD45a gene, but I didn't get clear bands, even when I changed the following conditions:
- High voltage (100V) to low voltage (80V)
- Gel concentration from 10% to 8%
I used oral cancer cells, but the results are so bad.
Maybe Your antibody is not specific enough or the concentration (of GADD45a or the antibody) is too high.
Hi Farhan,
Did you checked if there are any isoforms for this protein in this type of cancer?
Also maybe you should elongate the step in which you heat your protein mix to 95 degrees C (maybe 10 insted of 5 mins?) to make sure the proteins are all nicely unfold
What is the molecular weight of your of your protein. If its small M.W. protein use 15% gel instead of 8-10%. GADD45a is 18 or 36kDa protein if I am not wrong, use higher percentage gel to detect protein of this M.W. Try to use antibody which people used in published literature with positive controls. It could be possible that the cell line you are using have no endogenous GADD45a expression.
Hi Farhan,
1. What is the dilution of your secondary antibody? Use the least dilution possible.
2. After the incubation with secondary Ab dilution wash your blots extensively (7 times each 5 minutes) and also add tween20 to your wash buffer (0.1%).
3. If you still the see the non-specific bands. Then if the bands are smaller in size than your target then check whether there are known splice isoforms for your protein. If the band larger in size than your target then check whether it is exactly double the size of your target protein then it should be doublet.
4. If none of the above works then you should order antibody from some other vendor alternatively you can also play with the primary Ab dilution.
Good luck
Ram
How much protein are you running for the western blot? After your transfer method, do you see nice running of the protein on the membrane? What type of membrane do you use and how do you block? What do you dilute your primary and secondary antibodies in? If you use BSA and you are getting lots of background, try using milk instead. Running a positive control lysate for Gadd45 or purified Gadd45 protein might help show what you should look for under your running conditions.
i use 25 ug to 30 ug protein for western blott, i used antibody from abcam... 0.5 % skim milk in TBST for 1hr and 30min..while dilution of primary antibodies are 2ul/10000ul..after running the gel i check the bands with poncheous solution its seems okay..in comparison the marker bands are clear and intact on same blott.
Hi Farhan,
As I suspected your secondary Ab dilution is very high. Try the 1ul/25000 and 1ul/50000 dilutions that should get rid of many non-specific bands.
In my experience antibodies from AbCam are always of very good quality. Try the above suggestion I hope it solves your problem.
Good luck
Ram
Use High concentrated SDS gel 12% or so as your gene molecular weight is low and even low your dilution of 2AB 1:2000 to 1:4000 or as its recommended from suppliers.
Thank You
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