Homemade ABC kit
klenchin at macc.wisc.edu
Mon Jul 24 22:57:31 EST 1995
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.
(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
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.
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):
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
[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 isnt 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
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.
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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