What might be the reason for ghost bands in western blot?
1. I have used 0.5% skim milk overnight blocking
2. Primary antibody dilution 1:1000 3hrs rocking (3xwash PBST/ PBS) RT
3.Secondary antibody dilution 1:1000 (3xwash PBST/ PBS) 1 hr rocking RT
4. Protein concentration 100ug
5. ALP BUFFER 10MIN INCUBATION
6. NBT/BCIP-33ul/ 3.3ul+963.7ul alp buffer 10 min in dark
I am trying to get bands but i couldn't. suggest me some ideas
Hi, Asiya, have you checked the protein transfer to membrane witn PONCEAU??
I think that 100 µg of the pritein is very high, may be you have aprobelm with ntransfer.
Also, concehtration of the secondary antibody seems to be quite high.
Can you also mention more details. which primary antibody have you iused? Perhaps, you can show us at least one picture..
Good luck!
Ya thank you but i checked with ponceau its good only. i will try by decreasing protein and secondary antibody concentration. My primary antibody is Akt.
High Protein concentration causes ghost band...Ponceau detection limit is different than Western blots.
If they are trying for Akt (total), not P-Akt, then blocking with BSA will not help- if anything- milk produces cleaner blocking- at least I have found. I would say that its the concentration of the protein in the lysate that you are using- usually 20-30ug is enough. For the primary, I would suggest looking up the tech sheet for the recommended concentration- if there is no specific information on that one- look up papers that used primary from the same company and what dilutions they have used.
I use 30µg of protein.
In my blots, one hour RT or 4C overnight for blocking doesn't change the afinity of the antibody. I use 10% skim milk in PBS-T (0,1%).
Washes: 2x with PBS-T (0,1%) and 1x with PBS-T (0,5%)
Antibody: it depends on their concentration. Generally 1:1000 for primary and 1:10,000 for secondary.
i use 25 µg of protein, 3% skim milk, 1 :1000 primary antibody then wash in PBS 1x and PBST for 5 min. after that using secondary antibody 1:5000 then wash in PBS 1x and PBST for 5 min.
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