Hello everyone, I have a question concerning the blocking step in Western Blot : Does it make any difference, for the quality of detection, if I change the time of blocking? I used to work with one hour, but some people suggest 2 hours. Can someone please explain the difference or confirm whether is correct or not. Thank you in advance.
Hi there,
Blocking for more than 30 minutes at room temperature is not necessary (3% non fat milk in TBST 0.1%). From my experience, I've always preferred to probe overnight with the primary antibody at 4°C rather than to extent the blocking step.
Hi there,
Blocking for more than 30 minutes at room temperature is not necessary (3% non fat milk in TBST 0.1%). From my experience, I've always preferred to probe overnight with the primary antibody at 4°C rather than to extent the blocking step.
I agree to the previous answer, From my experience, I block my blot in 40C for 1 hours, and it work well.
blocking is a crucial step for western blotting... You need to optimize you blocking time.. Shorter blocking periods results in non specific binding whereas prolonged blocking leads to weak signals... In Our lab we prefer to block membrane with 3-5% BSA IN TBST for 2hours.
I agree with many others. Anywhere b/w 30mins to 1h at RT should be enough. Why do you want to switch for 2hrs if you were getting good results (presumably) for 1 hr blocking?
Is relative to blocking concentration buffer, but 1 hour usually is enough
Blocking steps usually can be manipulated depending on the signal intensity of your antibody and also the concentration of the same. If you are getting many non specific binding, increase the time of blocking, may be you can also do it twice- once before primary incubation and next before secondary incubation. I usually use 5% milk in 1X TBST. Hope this helps. All the best.
It really depend on your blocking reagent. For fat-free milk it takes 90mins and for superblocking reagent which purchased from Thermo takes 60min, both of them work well. Hope this helps you!
Hi,
the duration of your blocking depend on the quality of your antibodies. it could between 30min and 2h. The more background (non specific interaction) they create on the membrane, the longer you need to do the blocking. Considering that you have titrated your antibodies and do not use more that you need to detect the band of interest, if your membrane are quiet dark, it often because either you have not blocked long enough or you need to change your blocking agent (BSA instead of milk or vice-versa). Hope it help
If you use latest modified blocking device from merck, I noted it take really shorter time. Just pour blocking solution e.g milk to the place on the instrument and on the vaccum attached to the instrument. once the milk are suck out all. it is done. Milk can be mixed with antibodies. Super fast.
So depending on what reagents, what instrument, it decides how many minutes to run for this step.
It's interesting consider the size of your membrane, and also the amount of protein transfered to then that want to detect. I think it is enought a block time between 1-2 hours at room temperature, in a solution of 5% non-fat milk in TBST 1x.
As already mentionned by others, 30minutes-1h in 1xTBS-5% dry milk or 1x TBS-5% BSA at RT is enough... from my experience.
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