Elizabeth,

This isn't difficult, the two main secrets are 1) the water and 2) 
the composition of the tubes in which the conjugating is done.
1) 18 meg-ohm (though this may not be strictly necessary) Type I 
water is needed, but even that may not work. We have a Barnsted 
Nanopure that produces 18MOhm water, but it's not much good for this 
purpose, so we've been using a Millipore SuperQ system. The 
difference is that the Millipore gets it's cartidges changes 
quarterly whether the Ohms are down or not, and it can be dismantled 
and cleaned completely -- the Barnsted can't. I have used <18 MOhm 
water from the SuperQ successfully.
2) Plastic tubes are fine, but not all plastics. Polystyrene causes 
the conjugated gold-antibody to precipitate out on the tube. 
Polycarbonate seems to be best. Polyethylene (microfuge tubes) work 
for most antibodies.

Also, you need to know the pI of your antibody. pH the gold using 
K2CO3 (HCl if the pH is too high) *carefully!* a tiny little bit 
causes a big change in pH.
Make the gold slightly (0.1 or 0.2 pH units) more alkaline than the 
antibody pI.
Some antibodies and proteins are very sensitive to this step, and 
others aren't. IgGs usually aren't, and do fine at pH 7.2.
If needed, run a pH gradient: one concentration of antibody and 
different pHs, 0.5 units apart to start, then again at 0.1 units. But 
you don't want to do this unless the concentration needed is already 
known.

Do a concentration gradient: usually 0 to 10 microgram/milliliter is enough.
1) Use a volume of antibody about equal to 10% of the volume of 
colloidal gold, and keep 2 tubes without antibody (these are 
controls) -- add and mix.
2) Let stand about 5 minutes just to be sure (15 minutes is OK).
3) Add 10 to 15% by volume of saturated NaCl (10% NaCl is OK) -- add 
this to one control tube as well. the control tube should immediately 
turn blue, if not, something's wrong. Add nothing to the other 
control tube -- these are your color references. NOTE: *Na*Cl! 
Monovalent! The ability of a salt to cause the colloidal gold to 
aggregate (that's what the "turning-blue" is) is a function of the 
6th power of the valence, so only a tiny amount of a divalent ion or 
vanishingly tiny amount of a trivalent ion is needed to make the gold 
aggregate. This isn't good -- too hard to measure.
4) The lower concentration tubes should turn blue more - or - less 
immediately, but they can be left sitting for an hour to overnight in 
the 'frig to test for stability.
5) Look for the lowest concentration tube that stayed red, the color 
of the control tube with no salt. Use this concentration, maybe the 
next higher.
Purply is OK, sort-of. There will be some aggregates, but it will 
generally be useable, but not stable for any period. With some 
proteins, this purple-red color is unavoidable. But red the color of 
the nothing-added control tube is preferred.
6) Using this concentration, make up your colloidal antibody. Add 
water equal to about about 10% of the volume of gold to the antibody 
- gold mixture (this step can be skipped). If the mix must be stored, 
stabilizer like bovine serum albumin or polyethylene glycol can be 
added, but this should be avoided.
Spin. This depends on the antibody and size of gold. For 10nm gold, I 
use 30 minutes at 15,000 rpm. Refrigerate while spinning! Do in a 
'frig if using a micro-centrifuge, or a refrigerated centrifuge. (For 
other sizes, I don't remember -- I'm at home, and we're moving the 
lab as well. I might be able to find the information ...)
The conjugated gold-antibody with form a soft, loose pellet. Suck up 
this and dilute with your working buffer to an optical density of 0.5 
at 520 - 525 nm (wavelength depends upon particle size).
This should result in particles covered with one layer of protein, at 
a concentration of about 6 X 10^12 particles/mL. Maybe more -- again, 
I'm at home, and not remembering precisely. This concentration allows 
for a reasonable labelling time (a few minutes). 10^10, say, can take 
an hour or days to label.

Making the colloidal gold is easy and cheap as well. Easier than 
conjugating. Easier than conjugating. Let me know if you need any 
recipes for this.

Mind, some proteins are a *real* pain to conjugate. These are usually 
the real expensive ones that only come in minute amounts. (The 
concentration gradient can be done using microliter drops on 
Parafilm.)

Phil

Let me know if you have questions. These methods are in the 
literature, and there are several books available.

>Hi,
>
>     Does anyone have a protocol for conjugating colloidal gold to an
>antibody that they would like to share...or any info on the technique?
>
>Thanks
>Elizabeth

-- 
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Philip Oshel
Supervisor, AMFSC and BBPIC microscopy facilities
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706-1284

voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)
peoshel@facstaff.wisc.edu