Western Blot Background Troubleshooting

[molecule2005]

Hello,
I want to compile a good Western Blot Background Troubleshooting guide.

Does any one have experience in this area?


So far I have several problems of Western Blotting Background with solutions:

1) Too much secondary antibody used causing high background

Solution - use less secondary antibody (try 1:10,000 or more)

2) Wash time during washes (between antibodies etc) too short

Solution - increase your washing time

3) Wrong western blot membrane type used

Solution - use the right membrane type. For minimum background, try using PVDF membranes for western blot transfer.

4) Blocking Reagent (non-fat dry milk) is too diluted

Solution - Make sure you properly prepare FRESH nonfat dry milk (5% w/v) dissolved in TBST or suitable buffer such as PBS.

5) Contamination or Dirty membrane and/or blotting equipment

Solution - A) Use clean equipment, freshly prepared buffers, and new membranes.
B) Always avoid handling and touching membranes with bare
hands (even with gloves handle very little). Make sure you use gloves and forceps to handle western blot PVDF membranes.

please post your inputs here

[danfive]

My biggest tip for western is to make sure that your protein preparation is optimal, for mammalian cells I recommend CHAPS lysis buffer with incubation on ice. Also to know ahead of time if you want to reduce the proteins or run them in native state.

[bactin]

Hi
nice tips,neatly compiled.
Thanks for it.

[lovelyday713]

Hi there,

Here are a few more troubleshooting for western blots.

1) Bubbles

[lovelyday713]

sorry..
1) Bubbles
Sometimes bubbles are an issue. I use a roller for gels (biorad) that I use to roll over the nitrocellulose once put on the gel and then again on the whatman paper that goes over that, that makes sure that no bubbles are present. Also make sure that everything stays really wet and goes straight into the transfer tank (already filled with buffer) Drying out of the nitrocellulose can also result in bubbles and bad transfers.

2) Weak signals
Can be the result of too little primary or secondary antibodies and this needs to be titered out. Usually it's a primary that can go to less dilutions and not so much the secondary, but each case can be different.

3) Static signals on the film
These look like blobs everwhere. sometimes caused by static, to get a really nice looking blot, I take my probbed nitrocellulose, after putting ECL and place it in a plastic sheet protector. I then reduce the static by wiping down with a dryer sheet. And then finally put the film on to develop... removes static everytime!

These are all I can think of off the top of my head. I've been doing westerns for many years though and have all sorts of tips if the memories can be triggered!!

If you have an more specific problems, post them!

[danfive]

I also recommend using the Storm Scanner (GE) for image capture/blot analysis.

[rimapant]

I would like to know which dryer sheet you use. Paper towel,Kim wipes or special drying shee

[paramo]

1)weak signals
2)too strong signals ^_^
it`s hard to calibrate them, if u do blot not very often.

it`s normal, when unused developer (Kodak-for films) comes black?

[damertens1]

Hi Molecule 2005,

I really like your idea of a troubleshooting guide for Western Blot.
We have problems with blotchy Western Blots. It looks like some detergent / fat a.s.f. at some point is getting to the membrane.
We use semidry blotting, antibodies in milk, blocking in milk 3hrs RT, blocking & secondary ab in weighing plastic trays, hybridization primary ab in 50ml falcons.
Any suggestions would be greatly appreciated!
Thanks,
Daniel

[pabells]

Pierce has a pretty good tech support file on westerns. piercenet.com