I am working on autophagy.. trying to check the release of free GFP after autophagy by western blots.. but i am getting non specific bands.. how to avoid them and develop clean and neat blots ....?
Hello Naresh
There are many things to bear in mind to avoid non-specific bands while performing Western Blot.
1) Blocking of non-specific binding must be carried out with 3-5% BSA or non-fat dry milk for a period of 1 to 2 hrs.
2) Do not use the primary antibody at too high a concentration. Find the optimal concentration and try to use a more diluted primary antibody keeping the incubation period longer for better results.
3) Keep the secondary antibody dilution at 1:10000 to 1:20000.
4) Washing steps must be performed many times (5 mins) each.
5) Do not allow the membrane to become dry during the Western Blot procedure.
All the best.
Hi Naresh,
The three most important things are blocking, washing and an adequate antibodies concentration. If this can help you, for blocking I use 3% Nonidet NP-40 in PBS for 30 min, and wash three times the nitrocellulose membrane with PBS. Then, after each step of incubation with sera or antibodies, membranes are washed three times with 0.5 M NaCl, 2 mM Tris-HCl pH 7.4, 0.1% Tween 20. Regarding antibodies concentration perhaps you could try the assay wuth lower concentrations.
Hello Naresh
There are many things to bear in mind to avoid non-specific bands while performing Western Blot.
1) Blocking of non-specific binding must be carried out with 3-5% BSA or non-fat dry milk for a period of 1 to 2 hrs.
2) Do not use the primary antibody at too high a concentration. Find the optimal concentration and try to use a more diluted primary antibody keeping the incubation period longer for better results.
3) Keep the secondary antibody dilution at 1:10000 to 1:20000.
4) Washing steps must be performed many times (5 mins) each.
5) Do not allow the membrane to become dry during the Western Blot procedure.
All the best.
Thank you for your valuable suggestions i will implement them María Luisa Caballero
Dear Malcolm Nobre thank you for your suggestions i am doing all the steps mentioned by you but still getting some non specific bands Malcolm Nobre
Dear Naresh Kumar M ,
Sometimes it is difficult to avoid non-specific bands. You can try to use different blocking solution and fing the right one, and then dilut your antibody (primary and secondary) with different %% of blocking solution (milk or BSA or smth else).
One critical factor is the antibody itself. Some will give you non-specific bands no matter what you do:-( Cell Signalling products don't usually have that problem.
That being said, follow manufacturer's instructions for type of blocking protein (BSA or Milk), Buffer (TBS or PBS), and detergent (usually Tween 20 at 0.1%). I agree that lower concentration of primary @ 4ºC, overnight, usually solves many problems. Good luck!
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