Homemade ABC kit

Dima Klenchin klenchin at macc.wisc.edu
Mon Jul 24 22:57:31 EST 1995


Hi!

just happened to write it down today while sitting by a
painfully slow gel filtration. Hope it works for you.

Cause quite a few people requested this protocol, I wrote it 
down and sent out (Perhaps, too much details, but that never hurts). 
Since it now does not take anything from me, I decided to post it 
just in case if anyone will find it useful. 


Versatile sensitive non-radiocative blotting detection system. 
           (Home-made Avidin-Biotin-Complex kit).


There is nothing original below. Just a summary of our 
experience. Cookbook-like protocol for Westerns included in 
the end, too. 

I. Reagents.
	
(This is for Westerns only but the ABC preparation described 
below could be equally well used for nucl. acid detection.) 

	1. NeutraLite Avidin (Molecular Probes # A-2666, 5 mg, 
$78, said to be > 14 u/mg). It is decarbohydrated avidin. 
Supposed to be better than avidin because of lesser pI 
(~6.0) and better than streptavidin because does not have 
RGD sequence. The price is ~ same as avidin, < than 
streptavidin. Will be abbreviated as N-avi. 
	2. Biotynylated alkaline phosphatase (Sigma # P1318, 
1mg protein, $113.05, X-biotinylated at ~ 4 mol/mol, ~ 3000 
units/mg). I think, it's the highest sp. activity available at a 
reasonable price, but if same is found as XX-conjugate or 
with higher degree of biotinylation, this will be better. In 
the latter case, ABC titration (see below) is highly 
recommended. Will be abbreviated as bio-AP. 
	3. Any biotinylated protein (for example, X-bio-BSA, 
Sigma # 6048, 5 mg, $18.80). This is needed for ABC 
optimization only. 
	4. Biotinylated Goat Anti-Rabbit IgG (Sigma # B 8895, 
1ml, $56.40). This is NHS-biotin without linker arm. With X-
linker should be better (but only if the price is the same). 
They are absorbed with human IgG (supposed to decrease 
background, does not hurt as a precaution). Will be 
abbreviated as bio-IgG. If one has primary Ab raised in 
different animal - change secondary in accord. 
	5. Normal goat serum (Sigma # S 6898, 100 ml, $21.95). 
Should be dialized extensively against 4X2 l of TBS + azide. 
This is really optional - precaution to reduce background 
caused by secondary antibodies. If there is absolutely no 
need to reduce background - don't bother. 

II. Solutions.

	1. "TBST": 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, 
pH 8.0 (could be modified pretty much as you want in regard 
of salt, detergent, pH but absolutely no phosphate allowed).
	2a. General "blocking solution": 1-3% BSA in TBST (or 
5-10% dialized serum that does not interfere with either 1-
ry or 2-ry Ab; as one netter pointed out, Pierce's 
"SuperBlock" solution that is not expensive, is biotin free 
and is very good, could be just perfect). Milk is not good 
because of biotin content. 
	2b. Secondary Ab blocking solution ("serum block"): 5% 
goat serum in TBST. 
	3. "Antibody incubation solution": 1% BSA in TBST
	4. "Washing solution": 0.1% BSA in TBST (BSA or any 
other blocker is pretty much optional here). 

	5. ABC stocks: dissolve N-avi and bio-AP in 50% 
glycerol in TBS + azide to ~ 1 mg/ml. Store at -20C. (I 
usually aliqout them, freeze everything but one in liquid 
N2, thaw only once and keep at -20C). Alternatives: i) not 
to aliquote at all, or ii) to use Pierce"s "SuperFreeze" 
(the description sounds *very* good), to trust them, not to 
aliquote and sore at -20C hoping that it will provide > 1 
year stability (even with glycerol alone no detectable 
changes in ~ 2 months). 

III. ABC optimization

(Following is based on a chapter in Meth. Enzymol., v. 184, 
Avidin-Biotin Technology, don't have ref. on hands, but is 
easy to find). 

The idea of ABC is to detect any biotinylated substance 
using polymeric complex formed between polyvalent avidin and 
bio-protein. Obviously, there will be: i) optimal avi/bio 
ratio and ii) optimal absolute concentrations of reagents. 
This is found empirically using the following procedure. 

	1. Take 16 small membranes (NC for this is most 
convenient; we use pure NC 25 mm filters from Whatman that 
we buy for filter binding assays), prepare serial 1/3 
dilutions of biotinylated protein (like bio-BSA) in 1% BSA 
to cover the range from 10 ng/ul to 10 pg/ul. Load 1 ul of 
each on each membrane, let dry, block and incubate membranes 
for 30 min separately with a mixtures of equal volumes (NB: 
*mix quickly while vortexing*!) of different concentrations 
of N-avi and bio-AP (diluted with 1% BSA or whatever works 
best and does not have biotin; concentrations are given 
*before* mixing; after that, allow 30 min -*important*- for 
the complex to form before adding it to the membrane): 

				bio-AP, units/ml

                     1         2         4              8

		1    x         x         x 		x

N-avi, ug/ml    2    x         x         x 		x

		4    x         x         x 		x

		8    x         x         x		x

	2. Develop with whatever you like (BCIP/NBT, Naphthol-
phosphate/azo, chemiluminiscent). Choose the combination 
with the best signal/noise ratio and use it from now on for 
everything you do. (Actually, we chose 2 different: the one 
that is most economical but still sensitive enough for daily 
life, and the one that is most sensitive but requires more 
reagents - for critical cases; with the above reagents it 
corresponds to ~ 1ug/1u and 4ug/4u, respectively). 

	Note: for this dot blot optimization 35 mm Petri dishes 
are very handy and economical. Absolute sensitivity at this 
point does not matter so development time could be either 2 
or 20 min. 

IV. Titration of secondary antibodies. 

Exactly the same dot blot procedure: 1 ul serial dilutions 
of normal rabbit serum (1:1000 - 1:500000) or any rabbit IgG 
are loaded on the filters, filters are blocked in 5% goat 
serum for 60 min, washed, incubated for 30 min with 
appropriate dilutions of secondary bio-IgG (1:1000 - 1:20000 
in case of the above stuff from Sigma ), washed, incubated 
for 30 min with the optimal N-avi/bio-AP mixture, washed, 
developed. Choose whatever you find the best. 

V. Titration of primary antibodies ideally (as with any 
blot) should be done too. This time it should be real life - 
SDS gel, transfer, etc. 

VI. Cookbook recipe
    ^^^^^^^^^^^^^^^
This should work pretty well with the reagents listed above 
even without optimization. ABC fine tuning is always 
possible (see above) and everything about troubleshooting 
Westerns (time vs dilution vs temperature, blocking agent, salt 
and detergent conc., etc etc) is applicable here to the 
same extent. 

[Everything is in TBST]

1. Wash blotted Immobilon-P membrane in TBST (2x2 min)
2. Block for 2 hr with 3% BSA
3. Bind primary Ab for 30 min in 1% BSA in (<~ 10 ml in a 
plastic cover from 200 ul tips rack or smth like that)
4. Wash at least 3x10 min in > 20 ml of 0.1% BSA 
5. Bind bio-IgG (1:3000 normally, 1:15000 when high 
sensitivity isn’t important) for 30 min in 5% goat serum
6. Wash as above
7. Prepare 5 ml of 1 ug/ml N-avi in 1% BSA, 5 ml of 1 
unit/ml bio-AP. Mix them on vortex at high speed, leave for 
30 min at RT. (Note: use 4ug/4units when sensitivity matters 
so much).
8. Incubate blot with Avidin-Biotin-Complex for 30 min. 
9. Wash as above.
10. Develop the blot 

(I strongly prefer Naphthol-AS-E-Phosphate/Fast Violet color 
development as described in Anal. Biochem., 1990, 190:254, 
but I will definitely switch to chemiluminiscence as soon as 
screens similar to PhosphoImager appear nearby). 

If everything is done properly, detection of ~ 1 pg of 
protein should not be a problem. 


Disclaimer
~~~~~~~~~~

The following is a protocol that works fine for us and I tried 
to describe it in sufficient details to the best of my abilities. 
I, however, must say that I do not guarantee exact same results 
and am not responsible for any failures. No connections with 
any of the companies mentioned. 


- Dima Klenchin, University of Wisconsin at Madison

klenchin at macc.wisc.edu







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