Western Blots: no signal - low or no detection signal (Jun/16/2005 )

Hello everyone,
I'm trying to figure out what could possibly be wrong in my procedure because recently I have developed at 6 blots and got no signal at all even after over night exposure.

Procedure I follow:

1. Blocking in 5% milk (PBS-T) for 2 hrs @ RT
2. Primary Ab incubation o/n @ 4C in milk+(PBS-T)
3. Wash 3x30 min in PBS-T
4. Secondary Ab incubation 2hrs (milk+PBS-T)
5. Wash 3 x 30min
6. Developing
7. Exposure

All reagents are fine. Does too much wash significantly reduce the signal????

Why you really wash so long?
I wash 3 times 5 minutes

1ab I incube 1h, 2nd, 0.5 h

If you have no signal, maybe you reagents are not ok. have you dark background ater developing? With what substrate you develope?
What are your 1st and 2nd ab, maybe their concentrations are wrong

Are you shure, that your protein was properly translated, with no frameshifting?


Procedure I follow:

1. Blocking in 5% milk (PBS-T) for 2 hrs @ RT
2. Primary Ab incubation o/n @ 4C in milk+(PBS-T)
3. Wash 3x30 min in PBS-T
4. Secondary Ab incubation 2hrs (milk+PBS-T)
5. Wash 3 x 30min
6. Developing
7. Exposure

All reagents are fine. Does too much wash significantly reduce the signal????

hi
do you have positive controls for your experiment? did it work?

I guess I have to start over and remake all the buffers........ Thank you.

The washes do seem a bit long, but I'm unsure that they actually cause you to lose signal (only precious time).

I have experienced loss of signal when transfer to the nitrocellulose membrane was done using old(er) transfer buffer. We use fresh Towbyn transfer buffer for each run, but for reasons unknown the transfers are most efficient in the newest stock. This can easily be seen by looking at the color intensity of the ladder on the nitrocellulose. I assume your ladder transfered normally?

Also, you might consider using a reagent like SuperSignal or similar in visualizing your bands. These high-intensity reagents are apparently much more stable than the normal ECL peroxide-based reagents, and will also give off greater light than ECL. SuperSignal is not useful when the western is for a highly-abundant protein, since overexposure can occur much faster.

Ladder transfered perfectly. Bright colors. I don't doubt the transfer - I think my problem lays somewhere between blocking and development/exposure. Which company manufectures SupreSignal?? Is it also Amersham?

hi. do you do a ponceau stain to cehck that your proteins have transfered properly? my washes are normally 4x 10 minutes. and how long do you do the western transfer for? that might also be a factor. hopefully your primary antibody is still active too.

Check to make sure your ECL reagents didn't go bad. Mix 0.5ml of each in an eppendorf tube. Vortex. Add 1ul of your secondary antibody and revortex. It should turn brown.

you mentioned "no signal at all",
can you see your membrane or membrane edge after overnight exposure (background signal)? if not, check your substrate and secondary antibody, otherwise introduce a positive control next time.