Primary antibodies, Incubation time, western blotting, ELISA, SDS-PAGE
Hi Yogendra
As previously mentioned it will really depends upon the antibody of your choice. If it's specific, freshly prepared and in proper concentration (you need to work out what concentration is optimal), 1 hour incubation at RT should produce enough signal for detection with either chemiluminescent or colorimetric approach. Please make sure you incubate with agitation.
Best wishes.
2 hrs. This time is enough to obtain a good signal of the protein of interest. However, you should follow the recommendations of the manufacturer in order to obtain reliable results.
Generally speaking, you can use the recommendations (if any) that come with the technical bulletin for your specific antibody (sometimes antibodies belong to a kit but are sold separately, so you check the kit as well). Otherwise, overnight at 4oC is a common choice. We do this for primary Ab in both ELISA and western blot.
I usually incubate over night at 4°C for western blot but in the end all depends on the technical recommendations for each antibody.
Usually i incubate overnight at 4 C on a shaker. Gives very good signal
I m very thankful to u all for giving ur best but I m thinking why overnight its too much time ...if we need result quick generally wait for 2-3days ??
I would recommend you to use technical bulletin for particular antibody to determine incubation time.
And we do that in our lab but, in general practice we incubate for 1 hour @ room temperature or overnight @ 4 degree Celcius on a shaker.
I hope that helps.
Anywhere between 2 hours to overnight at 4C, is usually good enough
Usually i incubate for 1 hour @ room temperature or overnight @ 4 degree Celcius on a shaker this time is enough to obtain a good signal of the protein of interest. However, you should follow the recommendations of the manufacturer in order to obtain reliable results.
Hi Yogendra
As previously mentioned it will really depends upon the antibody of your choice. If it's specific, freshly prepared and in proper concentration (you need to work out what concentration is optimal), 1 hour incubation at RT should produce enough signal for detection with either chemiluminescent or colorimetric approach. Please make sure you incubate with agitation.
Best wishes.
Hi Yogendra,
It really depends on your primaries and your samples. I would suggest you to do an overnight incubation at cold room with suggested dilution that listed in datasheet. Forgot to mention, I normally would put 50ug if protein lysate in a well to test my primaries. If the signal you expected did not show up, I would increase amount of protein lysate in each well, and hybridize primaries at room temp for a couple of hours. I even have an antibody that needs to hybridize at room temp overnight to get the best signal noise ratio. Hope this answer will help!
Good luck!
I agree with both of them. You can load differnt amount of protein in the same gel (10-100) and then go for 4C overnight incubation. This will help to determine the specificity of your antibody.
Hi,
As mentioned before, it depends on the Ab, the dilution, your protein etc. I have tried dilutions up to 1:5000 with incubations of 3h@RT or ON4C.
Good luck!
1-3 h at room temperature or Overnight at 4 degree would be fine. Use shaker at 15-20 RPM
I'd recommend overnight incubation at 4 degrees but you have to figure out the optimal dilition of your primary antibody (usually 1:500 - 1:1000) diluted in 5% BSA in PBS-t with 0.02% sodium azide.
good luck
incubate for 2 hours at room temp or 18 - 24 hours at 4 degrees celsius with gentle agitation (shaker low rpm).
You need to optimise the antibody dilution. In general it is around 1H or 1H3Omin.
In western blot usually we either incubate the primary antibody ( overnight at 4 degrees ) or 1-1.5 hours ( at room temperature) both gave me good results
The incubation of the primary antibody is between 1h and 1H30 min at room temperature.
It depends. However you can go either 3 hour RT or overnight at 4 degree .
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