i am doing western blot for my research study and i am getting the black spots on the membrane after developing which indicate the binding of first anti-body to skim milk. how can i eliminate this problem.
Hi Imdad,
There are some points that you may consider for troubleshooting this issue:
(1) Use a freshly prepared blocking buffer and make sure to keep the blocking buffer on slow shaker for an hour or so (before use) for completely dissolving the milk powder.
(2) Clean the buffer containers well to avoid any chances of bacterial contamination which often results in dotted blots, especially when the primary or secondary antibodies show cross-reactivity with bacterial proteins. It is common when using the same container again and again or when re-using the blocking buffer itself.
(3) HRP aggregates in the secondary antibody are another factor which may lead to this issue. One may use 0.2 µm filter to remove any aggregates or try a quick spin to settle the aggregates before use.
(4) If none of these suggestions work, you may consider trying a different primary antibody.
You can visit Novus support page at http://www.novusbio.com/support.html for various troubleshooting suggestions! I hope this is helpful but please let me know if you need additional feedback in this regard.
Best Wishes
Subhash
Hi Imdad,
There are some points that you may consider for troubleshooting this issue:
(1) Use a freshly prepared blocking buffer and make sure to keep the blocking buffer on slow shaker for an hour or so (before use) for completely dissolving the milk powder.
(2) Clean the buffer containers well to avoid any chances of bacterial contamination which often results in dotted blots, especially when the primary or secondary antibodies show cross-reactivity with bacterial proteins. It is common when using the same container again and again or when re-using the blocking buffer itself.
(3) HRP aggregates in the secondary antibody are another factor which may lead to this issue. One may use 0.2 µm filter to remove any aggregates or try a quick spin to settle the aggregates before use.
(4) If none of these suggestions work, you may consider trying a different primary antibody.
You can visit Novus support page at http://www.novusbio.com/support.html for various troubleshooting suggestions! I hope this is helpful but please let me know if you need additional feedback in this regard.
Best Wishes
Subhash
You're very welcome Imdad. Good luck with your experiments and feel free to ask if you need any other help :-)
If you are getting spots in your western blots, you may do the following-
1. As already suggested, using freshly prepared skim milk and to avoid any kind of contamination, you may block it at 4 degree for more time. At room temperature chances of contamination is higher. Store the buffer in 4 degree for the time between you preparation and usage.
2. Wash the blocking buffer thoroughly before you add primary antibody. Remaining milk particles may cause trouble when you develop the blot.
3. It can be due to secondary antibodies also.So, prepare antibody dilutions in buffers which are fresh and properly prepared.
Hope it helps!
I agree with all the advice above. You could also try to use gelatin (1%) or BSA (1%) for blocking instead of milk.
Use 3% BSA from blocking instead and optimize your blocking time at the temperature you are carrying out your blocking step. If the problem persists you can increase the BSA concentration upto 5% but even that dsnt solves your problem, you would need to change your antibodies and get then from a better menufecturere.. Also try using antibody solution diluted to a little higher dilution than the recommended minimum dilution. All the best.
Dear Imdad,
I think your problem might be due to unsufficient washing steps. I make 5 washes after the primary and secondary antibody incubations with a solution of 5% skimmed milk and 0.1% Tween20 in PBS and black dots have never been a problem for me. For blocking (1 h) and antibody incubation I use 5% skimmed milk and 0.05% Tween20 in PBS.
Anyway, there are also other possibilities. Have you had this problem with only one antibody? Is it a new thing? Did you change any product of method? Do other people from your lab suffer the same issue?
lot of thanks for the sincere help and guidance, i will certainly consider the suggestions offered by you peoples.
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques