Incubating the filters two overnights with primary antibody., WESTERN BLOTTING

  • Thread starter mountain
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Main Question or Discussion Point

Is that okey that I incubate the filter with primary antibody two overnights before adding secondary antibody for detection? This is because I do not have the equipment to detect the signals in the weekend so I have to detect it on a Monday. Due to it I incubate the filter with primary antibody on Saturday till Monday morning and then detect the filter on Monday. Would this give me problems?
 

Answers and Replies

DocToxyn
Science Advisor
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I wouldn't suggest doing this, you will probably end up increasing non-specific binding and thus will limit your detection. I would block the membrane with standard blocking solution (milk, BSA, whatever you use), rinse in TBST (or your other favorite buffer) and then seal it well in plastic wrap and store at 4oC until you can do the primary for the amount of time called for in your protocol. So you could do it Sunday night and then hit it with secondary and whatever detection system you are using on Monday.
 

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