Hi all,
I loaded 40 ug of HT-29 cell line protein, and I couldn't find my target band. But when I incubated it with the housekeeping gene, while detecting it showed two bands- faint bands for both-, one for GAPDH, and the other seemed like my target because it is in the size I was looking for.
NOTE, antibodies are cloned within two different hosts. one rabbit and the other is a mouse.
The question is, if my protein concentration is low, is it possible to increase primary Ab incubation time?
Thanks.
An hour is usually enough for the antibodies to bind.
What does a protein ladder look like? Maybe you have problems with the transfer?
Also try setting a longer membrane detection time in the gel documentation system.
Hi Дарья Коломийцева
Thanks for the reply, my ladder is good and all bands were successfully transferred. I think the problem is the low concentration I loaded since I used nanodrop for the quantification after I was using BCA.
about the longer detection time, i did this step but whenever the time increases bands become lighter!
Have a great day!
When I was trying to spot errors in my work, I came across information that light stripes in place of proteins meant that the concentration of antibodies was chosen incorrectly. But if you are sure that there is not enough protein, then simply load it to the maximum, without dilution, or put a series of dilutions in 1 gel, you will understand which one you need.
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