Immunochemistry - HELP! (Aug/12/2007 )
Hi, I'm pretty new at this so forgive me if my questions are dumb.
I'm trying to understand the principles behind Immunochemistry in Western Blotting.
So I was wondering if any of you could give me some feedback whether or not my observations are right or wrong.
Here goes:
Proteins, have gone through SDS-Page and Electroblotting.
Immunochemistry:
1. Membranes were blocked in TS (Tris-NaCl) buffer for 60 minutes at room temperature.
(This was done to block non-specific sites and thereby minimize non-specific binding of the detecting antibody)
2. Membranes were then incubated overnight at 4°C in primary antibodies diluted in a blocking solution.
(The primary antibody binds to the target protein of interest)
3. The membranes were then washed in distilled water
(To remove unbound probes)
4. The membranes were then incubated in secondary antibodies conjugated to HRP diluted in a blocking solution for 90 minutes at room temperature.
(Secondary antibody recognizes and binds to the primary antibody and visualizes the labeled protein with HRP)
5. Membranes were again washed
(To remove unbound probes)
6. The membranes were washed for 15 minutes in TS buffer containing 0.05 % Tween-20 and then washed 3x15 minutes in pure TS buffer.
Why? What does Tween-20 do precisely? Is it a kind of detergent that washes away "noise"?
7. The membranes were washed for 5 minutes in distilled water and prepared for visualization on film.
Questions:
What's the difference between 10%, 20% Gel in SDS-Page?
What's the difference between Monoclonal and Polyclonal antibodies?
Are the proteins on the membrane back to it's "natural form" when probing with antibodies?
Thank you in advance.
Cheers
Mifunes
(This was done to block non-specific sites and thereby minimize non-specific binding of the detecting antibody)
That's strange... Blocking buffer should contain some protein (eg. skimmed powdered milk, gelatin, BSA) or detergent (eg. Tween-20) in order to block.
(To remove unbound probes)
6. The membranes were washed for 15 minutes in TS buffer containing 0.05 % Tween-20 and then washed 3x15 minutes in pure TS buffer.
Why? What does Tween-20 do precisely? Is it a kind of detergent that washes away "noise"?
As good as I know you can skip step 5. As for tween-20 you're correct - it's a detergent and it's used to wash away "noise" ie. antibodies non-specifically bound to membrane and proteins.
They have different pore size and that gives different separation range, linearity range etc. Big proteins may not even enter high-precentage gels, while movement of small proteins through low-percentage gels won't be much retarded (poor separation).
Agree, the blocking solution (in step 1, 2 and 4) should have proteins and detergent for it to BLOCK.
I would not skip step 5.
Have never done washing with distilled water (step 3, 6, 7), I always wash with PBS+Tween20
About mono and polyclonal antibodies, in short (note that this is just generally speaking):
Monoclonal antibody: recognize single epitope --> more specific, less sensitive
Polyclonal antibody: multiple epitopes --> less specific, more sensitive
You can search out more about these antibodies and their uses in the Immunology and histology forum.
The proteins on the membrane will be like what they are in the gel. If you are running SDS, then no, they don't 'come back' to their native folding as long as the SDS still bound to them. That is why some antibodies work well for WB (usually recognize epitopes in the 'denatured' of 'straight' protein) but not for IF ('natural' protein) and vice versa. This all depends on epitope though.
Step 5 and 6 are both washin so what's the difference?
1st Q: What's the difference between 10%, 20% Gel in SDS-Page?
Difference is the poresize of the gel. the higher the % the smaller the pores. high % is for resolution of small proteins in SDS-PAGE and vice versa.
(ranges could be found in every textbook)
2nd Q: Are the proteins on the membrane back to it's "natural form" when probing with antibodies?
normally the proteins are denatured during treatment with SDS, Heat, MercaptoEtOH...; which means DENATURING conditions ("non-native"). in some special cases NATIVE PAGE is performed, e.g. when some want to check interactions (protein-subunits), modifications...
Sorry, what I actually meant is that I will do step 5, washing the membrane (here did not say in what so for me I wash in PBS/Tween20) and ignore altogether the complicated step 6.
Thank you so much for all the replies. It helped a lot. 