Indirect Fluorescence problem - (Oct/20/2010 )

Hi everyone,
I just started using IF to localize a soluble protein in the cell. The problem now is I always get strong signal on the peripheral of the cell, nothing inside the cell. This protein is definitely a chloroplast protein within the cell.
Here is my protocol:
1. Spin cells down, resuspend in the residue medium;
2. Affix to poly lysine slide, air dry for 20min.
3. Fix with 4% formaldehyde for 40min.
4. Three washes with PBS, permeablize the cell with 0.5% triton-100 for 10min, and methanol at -20C for 10min.
5. Dry the slides, and rehydrated with PBS for 5min.

6. Blocking: 2% BSA in PBS for 1 hour;
1st Ab: 1:100 overnight 4C or 1 hour RT; wash with PBS-T 6 times, 5min.
2nd Ab: 1: 500 or 1:800 1 hr in dark; wash; (Alexa488 goat anti-rabbit )
7. Observe.


My control is :
mutant without this protein; but still shows strong signal on the peripheral of the cell;
incubate with only the 2nd Ab; weak speckles of signal on the peripheral of the cell;


Any help and suggestions will be appreciated!

It sounds like your antibody cross-reacts with something on the cell membrane/wall and/or potentially you have a permeabilisation problem - you could try stronger detergents and a shorter fixing time to help solve these.

It may also be that your antibody doesn't detect native protein... have a look at the data sheet for it (if it is commercial) it should say applications that it has been validated for.

bob1 on Wed Oct 20 23:20:04 2010 said:

Thanks a lot for your input!

According to the EM pictures, the antibody does have some crossreaction with something on the plasma membrane, but 99% of the gold particles are in the right position.
I've tried tween-20 and triton x100 0.5%-1%.....Is there a feasible way to test the permeability of the cell, but bypassing the expensive 2nd Ab incubation?....

if the ab can be used in EM, it should be good for IF too...i guess....
thanks a lot again!