Dear Rouaa Shaheen

The best incubation time is often unknown in western blotting, and the timing of incubation could be performed to help determine maximum sensitivity for individual antibodies, in addition to titrating antibody concentrations. Also, antibody providers assist investigators by including timing data for maximum sensitivity in datasheets for each antibody.

The common protocol that you find on the internet (shorter incubation time) i.e., incubating for one hour” or “incubate overnight” in Western Blotting may appear to be insufficient to reach the maximum sensitivity that primary Abs and secondary Abs could provide in some cases.

Also, if you use the protocol followed by your lab mates i.e., incubating primary antibodies for 2-3 days at 4 degrees Celsius and secondary antibodies for 1 day (overnight) at 4 degree C may result in increased background and there is a probability of loosing signal strength over time.

So, I would prefer you decide on the timing of incubation for the antibodies. I am attaching an article for your reference that may help you in this regard.

Article Prolonged Incubation and Stacked Film Exposure Improve Sensi...

Best.

Hi Rouaa Shaheen

I perfectly agree with Malcolm Nobre, the ideal time of antibody incubation can vary from lab to lab depending on various protocols and reagents used in western blotting.

* You can enquire the ideal antibody incubation time from the distributors or application scientist from whom you have purchased the same antibodies. As the conditions would have been optimized by them. It is ideally advised to perform the same in our lab before confirming the results.

* Ideally in our lab we keep primary antibody incubation for around 8-12hrs in 4 degree refrigerator while secondary incubation for 1hr at room temp. The same time may differ for a few antibodies.

Best of luck

Hope it helps.

Thanks

Samir Ranjan

Dear Rouaa Shaheen

We don't know the reason behind incubating primary antibodies for 2-3 days and secondary antibodies for 1 day (overnight) at 4 degree C by your lab member, its better you asked them explanation behind doing that.

1. Most of the antibody mention the ideal condition in the data sheets.

2. Generally, 1h at RT or overnight at 4 degree for the primary antibody and 1h at RT for secondary antibody incubation is being followed throughout the worldwide but again it may vary antibody to antibody and lab to lab.

https://www.abcam.com/protocols/general-western-blot-protocol

All the best

Thanks

Pankaj

In my lab incubate primary antibodies for 1 day overnight at 4 degree C and secondary antibodies for 2-3 hrs at room temp. before band detection.

Pankaj Kumar Malcolm Nobre Samir Ranjan Panda

Do you know why the band little curve in the end of gel ? Through I run 25mA and 10% Gel.

Hi Nazmul Islam

l run with fixed voltage not current!

first at 50V for 10 min to settle down the protein, then at 100 V for 1.5-2hr.

The running time can be different based on the %age gel!

Pankaj Kumar

Thank you. How about the 15% Gel ,how much voltage do you use normally ?

Nazmul Islam

I keep the voltage condition same for any % gel, only running time varies! You can run till dye-front reaches to the bottom of the gel.