Hi all,
i have been doing western blotting for a while now. Got good results when i was a graduate student. But somehow in my new job, i have no luck! So i checked the lysate i use, is ok with sufficient protein concentration. I load 20g of protein per well. after transfer (nitrocellulose) i see that there are bands with ponceau. then i block it with milk, add primary antibody(1:1000) , secondary antibody (1:1000) adn develop the membrane for 10 minutes and expose for upto 40 minutes. still i dont get the bands or if i do get them i get at different molecular weight.

I have been using fresh lysate and other people in my lab got bands with same antibodies, same lysate (positive control).

Obviously i am doing soemthing wrong but i cant understand what? Please help !!! I am desperate.
Pinal

Hi Pinal,

Can you clarify a few things?
1. Did someone in your lab actually detect the correct bands with your lysates? or was a previous lysate for the positive control?

2. do you have western markers on your blot such as Magic Mark? these help to determine where things went wrong

3. how long is your primary on the blot? and what temprature?

4. what type of protein are trying to detect?

just trying to get some ideas before helping you trouble shoot

i usually back up and trouble shoot each step of the blot

1. is your protein of interest degraded quickly or actually in the lysate?
2. you already know you got good transfer of protein
3. you dont know if the primary is working (western markers will answer)
4. you dont know if the secondary is working (dot blot is good place to start)
5. is the developer working correctly?

Just some initial thoughts

Rb

Are you using the same secondary antibody as the other people in your lab? I know I've grabbed the wrong bottle once or twice and it's been a goat anti-rabbit instead of the intended goat anti-mouse!

Also, do your colleagues also use the primary ab at a 1:1000 concentration?

The only other thing that's happened to me is that with very small proteins (6-8 Kd) they have a habit of either running off of the gel before the blot or through the membrane and out the other side during the blot!
Shorter electrophoresis and blotting was the answer.

things I would check, assuming 1st antibody, 2nd antibody, detection reagent are all working.

1. 1st antibody, is the dilution rate right, I used to do 1:5000 to 1:10000 in stead of the producer's suggests of 1:1000 to 1:3000 to get better results, in most cases, 1:1000 is too too strong for western (for commercial antibody)

2. is the 2nd matching the 1st antibody

3. is the detection method matching the 2nd antibody

good luck

Hi all
thank you all for your suggestions .
I looked into all of these aspects nad heres what i found...

when i used HRP antibody , i was using it with milk buffer which contained azide. I found out that the azide will inactivate the secondary antibody. So i am reprobing my blots to see if i get some results after removing azide.

will keep u posted.
Thanks for your help
Pinal

yes, you have got your answer right, azide does inactivate HRP.
I hope u are getting your bands now.