I've done Western blot analysis of Notch 4 proteins obtained from normal and carcinoma cell lines. Transfer is complete onto NC membrane. I've prepared new antibodies with dilutions 1:750. I tried with BSA and skimmed milk but unfortunately didnt get any bands. Could anyone help me out?
The procedure i've employed
1) Transfer done using a semi dry blot.
2) Blocking for 2 hrs(tried with both BSA & skimmed milk)
3) 3 times wash with TBST buffer for 5 mins each.
4) overnight incubation with primary antibodies.
5) 3 times wash with TBST buffer for 5 mins each.
6) 2 hrs incubation with anti-goat hrp linked secondary antibodies.
7) 3 times wash with TBST buffer for 5 mins each.
8) Add DAB in dark & check for bands.
the basic suggestion to solve this problem
use wet transfer to ensure all proteins have been blotted to ur membrane, as semidry may be not so efficient (u can check the transfer efficiency by staining the gel itself after transfer).
Try to increase the 1ry Abs conc (1:200).
Try to increase the loading protein conc
Did you check for protein concentration? I agree with Shimaa that wet transfer is more efficient than semi-dry. Did you use a ladder and can you see the ladder after adding DAB?
Thank you all, i did check for protein concentration using Bradford assay. I loaded 50 micrograms in each well. No bands were seen in the marker after adding DAB.
Hello Anurag:
Couple of things to check:
1. Protein conc as Nur suggested (I follow BCA, it should not be too diluted) . 2 Your gel, how fresh it is. 3. Transfer in a wet system rather than semi-dry. 4. Activate the membrane with methanol before transfer. I usually activate the PVDF membrane for couple of minutes and then put it in the transfer buffer before transfer. 5. Check staining with Paunceau as Thomas suggested to make sure everything got transferred. Sometimes I stain the discarded gel with Coomassie blue which gives me some idea whether my transfer was good. If needed extend the time of transfer from just only 2 hrs to longer to get them transferred (in case of longer transfer you can move the unit to a cold box so the chamber does not get heated up. 6. Change the blocking from milk/BSA to just only Tween 20 for an hour and then add the primary (try a couple of different conc of the primary before the actual run). Incubation with primary overnight is helpful. Then follow the rest as you are doing.
Hope it helps.
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