philman

Ok, I have a couple of questions about using conjugated antibodies in both Western Blotting and immunoprecipitation.

We study a cell surface protein, and use a primary antibody followed by a FITC conjugated secondary antibody to cross-link the protein on the cell surface, causing it to form clusters that we can then see using immunofluorescent microscope. In order to provide consistency, I want to use the same two antibodies on the cells we are using for western blots, to investigate the proteins and pathways activated upon the surface protein cross-linking.

I have used this on western blots before, and not noticed any additional staining on the ECL film that I was worried may occur with the FITC fluorophore, so Iam not worried about it affecting that anymore.

What I am more worried about is the immunoprecipitation experiments.


1-Firstly, will cross-linking of the surface protein with two antibodies (A-primary mouse anti-human, and B-secondary goat anti-mouse) interfere with the use of a third antibody (C-mouse anti-human, different antibody to A) in the immunoprecipitation? Or will the whole complex bind to the Protein-G beads anyway due to the previous two antibodies used?

2-Secondly, The protocol states to use an isotype control antibody to 'clear' the lysates of stuff that may bind non-specifically to the protein-G bead/antibody complex. The only control antibodies we have in the fridge are conjugated ones, to FITC, PE, etc, for use in FACS controls. Would it be ok to use a conjugated control antibody in these experiments, or would the conjugation get in the way of the epitopes required to bind protein G?

Thanks, and thanks to any people around this early in the year to answer!

cold air wouldn't kill bacteria but it would slow their growth I would have thought. but then with cold air you have the problem of condensation occurring in areas not directly under the air flow, which bacteria would love to sit and reproduce in, think legionnaires disease, famously found in air conditioning systems.

The most efficient way to get sterile air would be to pass it through a filter, the same way the air in category 1 and 2 hoods is sterilised.

The most effective killing method would not be hot dry air, but hot wet air, dry heat is less effective at killing than wet heat, the hot steam can penetrate the aqueous capsules and cell walls that bacteria surround themselves with much better than dry air, hence why autoclaving (around 125 degrees C steam at high pressure for at least 20-30 minutes to allow full penetration) is the preferred method for sterilisation in most circumstances.

And yes everything depends on the species of bacteria, name an environment, you will be able to find a bacteria somewhere in the world that can survive it somehow! Common bacteria are usually killed by autoclaving or standard disinfectant chemicals. In general spore producing bacteria can survive much worse environments than other bacteria

If you're that worried you could just get some more media and combine the two together to double the volume and create a 1% solution (remembering to add extra FCS to keep that at 10%)

The general concentration equation is:

C1*V1=C2*V2

Where C is the concentration and V is the volume, 1 refers to starting conc/volume and 2 refers to the finishing volume. Basically it means that the concentration of the reagent multiplied by it's volume is always the same.

So for your question the starting concentration is 10% (so that is 0.1) and the end concentration is 0.3% (so that is 0.003) The end volume is 10ml

So in the equation we get:

0.1 x V1 = 0.003 x 10ml = 0.03

V1 = 0.03/0.1 = 0.3ml = 300ul

So you'd need to add 300ul of your 10% solution to 9700ul (about 10ml) of your diluent.

This is probably unhelpful but the first thing that came to mind upon seeing the full sized image was an Alien Facehugger...