Do you have some BSA in you gold-conjugated secondary Ab solution

Why do you want to avoid it? It shows you the current stage of your colloidal dispersion. If you have a colloidal stable system you will see individual particles.

Centrifugation is recommended to remove clumps. However from your image most of the gold is clumped. Is it possible that the epitope is actually in clusters ? How old is your colloid - it might be worthwhile purchasing a fresh supply. I always had good results with aurion conjugates.

At least two factors effect the gold particle solution: the size of the gold particles, and the age of the solution. The bigger the gold particles, and the older the solution, the more the particles tend to cluster. So, as suggested by others, you can try a fresh gold particle batch. I would also recommend using an inert protein such as BSA in the labeling solution (up to 3%). I always spin my gold conjugated antibodies at full speed (appr. 15000 g) for 2-4 minutes before using them. The time how fast the gold particles will sediment depends on the size of the particles (big particles and clumps sediment fastest). And yes, it is also possible that the antigen you are labeling is actually localized in clusters. You can easily test this I suppose, for instance by labeling another proteins that is known to localize differently. Or just apply the secondary gold conjugate without any primary antibody. If the gold is still in clusters, it is out of date.

I agree with Eeva-Liisa,

you should simply do negative controls, as there are: -testing the colloid on a empty grid to see if tere are clumps in your solution, - do a control without primary antibody to test the false positive signal, - use the same labeling on the consecutive section. If there are multiple epitopes you will find the same labeling on the next section as well. To spin down clumps we use routinely a table top centrifuge for approx. 2 min.

If you use gold conjugates, BSA should be already in there.

the problem is that I can not do negative control in this case, because 15nm Gold particles are conjugated directly to the primary Ab.

A quick and easy way to test your gold-conjugated reagent is to observe it directly deposited (at the corresponding dilution, or even concentrated as it is) on a EM grid (with a support membrane; formvar or collodion or carbon, etc), as it is done with negative staining (but without the staining agent).

You could also test your colloidal gold by analytical centrifugation.

So, I was thinking if I could remove Gold conjugates from primary Ab by centrifugation at certain speed and then use different gold solution to re-conjugate by myself to primary Ab. Cause, I think that company (where I bought this Gold conjugated Primary Ab from) used OLD/out of date gold solution.

So, how fast I need to spin (rpm, g) my primary Ab in order to remove conjugated gold particles ?

Good Morning,

dear David, regarding your last sentence with question above, may I point you to the reply given by

Eeva-Liisa Eskelinen • Helsingin yliopisto (4 days ago)?:

< full speed (up to 15,000g) for 2-4 minutes before use>

Notice:

The necessary g-force to be chosen depending on gold particle size, the achievable g-force depending on the type and radius of rotor used with your centrifuge....

cf. e. g. from: http://www.mpbio.com/detailed_info.php?family_key=02154012

“….The gold conjugate must be purified from excess antibody and any small clusters removed before concentrating and storing.

1. Spin the gold conjugate at the following speeds according to gold particle size:

5 nm 45,000 g for 1 hour @ +4°C

10 nm 25,000 g for 1 hour @ +4°C

15 nm 15,000 g for 1 hour @ +4°C

20 nm 8,000 g for 1 hour @ +4°C

These speeds and times are approximate and must be determined experimentally.

2. The gold conjugate will form a loose precipitate at the bottom of the tube. Discard the clear supernatant and resuspend the pellet into 2 ml of buffer A.

3. Prepare a 10 - 30% glycerol gradient (10 ml) in buffer A and carefully load the suspended conjugate onto the top.

4. Spin the gradient in a swing out rotor as follows according to particle size:

5 nm 125,000 g for 45 min. @ +4°C

10 nm 50,000 g for 45 min. @ +4°C

15 nm 15,000 g for 45 min. @ +4°C

20 nm 12,000 g for 45 min. @ +4°C

These speeds and times are approximate and must be determined experimentally.”

NB:

Googleing for < centrifugation of gold conjugated antibodies > or < Preparing Antibody-conjugated Colloidal Gold > you’ll get a lot of positive and results, esp. first results page.

Best wishes and good luck, Wolfgang MUSS PhD, Salzburg, Austria

I refer to Davids question: "So, I was thinking if I could remove Gold conjugates from primary Ab by centrifugation at certain speed"

Just to clarify one point: Wofgangs protocol above will separate the antibody-conjugated gold particles from the UNBOUND antibody (but not the conjugated antibody from the gold). If you wish to prepare an antibody-gold conjugate yourself, you should use a fresh gold colloid. Preparing gold particles of a certain size is not very complicated. It is also possible to buy unconjugated gold particles, but then you need to make sure that they are still suitable for conjugation (that is, not stabilized by a saturating concentration of inert protein like BSA).