I blocked with skimmed milk, did the 1ry Abs incubation overnight at 4C.
I have used two Abs (two membrane) for detection of integrin b1 using western blotting.
Washed 5 times with TBST, each is 10 min.
used secondary Abs 1:10000
Washed 5 times with TBST, each is 10 min.
I used the same secondary Abs for both primary Abs *anti-rat-HPR, visualize with HPR-substrate.
One of my membranes show completely black membrane using Li-cor as a scanning device.
The other one showed no spots, but rather it showed one positive band in one of marker places(Li-cor).
If I understood well, you tried two different antibodies for Integrin1b, but used the same anti-rat-HPR conjugated secondary antibody. When you scanned, you saw nothing on one membrane while the other showed up with a band for Integrin1b.
If my understanding is correct, is the species origin of both the primary antibodies used are same? Providing antibodies details will be helpful.
Thanks Vinod Nadella for ur reply.
I could not see any thing in both membranes, however one of them has positive band in the loading marker (cross reactivity) not a true integrin b1 band.
while the other one is completely black (no cross reactivity).
Both primary antibodies are rat integrin b1 but Abs would identify different epitopes in integrin b1. (Species origin are the same)
secondary Ab is the same one, anti-rat-HPR.
what I could understand from these results, that both 1ry Abs did not identify any integrin b1, however both are not black after scanning, since one of them has a cross reactivity while other did not show.
This is why I asked for further tips for blotting of integrin b1.
1-At first, make sure that the primary antibodies are selective to the protein target ( i.e. if protein source is human, antibodies to be anti-human). Because some antibodies are designed for recombinant proteins not native ones.
2- follow the manufacturer protocols for dilution, and if needed increase the antibodies concentrations.
3- Try not to denature your samples at 95 C (SDS-PAGE step), because some antibodies epitopes can't detect denatured protein target.
4- run SDS-PAGE at low voltage to avoid heating.
5- check from literature the expression levels of your protein, because the expression levels differs from cell to cell.
6- you may also try overnight blocking by skimmed milk at 4 C.
Good luck
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