What do you mean by appropriate buffer? Why are you running centrifugation after titration? Can you disperse the black spot using sonication? Centrifugation will always favor particle agglomeration. Reduce rcf or run dialysis.

thank you for your reply.

i meant a buffer that resists pH changes like borate or PBS, as I am going to set different pHs (pH=7-9.5). I do not do cetrifugation after titration, centrifugation just for changing buffer before and after conjugation. Black spots resists any types of resuspension.

If aggregation happens before or after functionalization, the nanoparticles are not stable enough to win over the attractive forces.

After centrifugation, resuspend particles by vortexing first. Cool to 4oC then use a sonicator probe. 2 cycles of 10 seconds at 40% Ampl should do it

If the particles aggregate after centrifigation you can use any sonicator you want and still won't be able to recover particles as single entities.

Thank you all for your answers.

there is no aggregation after conjugation but when I centrifuge samples a metallic spot appears at the bottom of micro-tube. I did not block my samples with BSA or PEG after conjugation, do you think it could be the reason? the other problem is that if I block the samples with BSA or PEG then i would not be able to use BSA for measuring the concentration of protein in supernatant?

It is essential to block the gold after conjugation. You can use PEG-20000. It should not interfere with protein assays especially the modified Lowry assay

I am using this protocol for physical conjugation but in the last step it aggregates. could you please tell me in which step I am making mistake:

1. gold particle of 20 nm in 1.5 tube at pH=PI of protein (GNPs are disolved in 0.1mMPBS)

2. 0.01 mg of protein (I dissolved protein in 0.01nM PBS but I do not know what pH i should use).

3. incubation

4. blocking with 10 w/v% BSA and incubation for 15min

5. centrifugation

6. re-suspending the pellets in ultra-pure water (when I use buffer, during centrifugation the red color of the sample sticks to the tube wall)

7. centrifugation and re-suspension in 0.1%w/v BSA (aggregation happens at this step)

does this mean no conjugation has happened?

thank you in advance

You should never use phosphate buffered saline (PBS) with gold particles. Avoid sodium chloride at all cost. Keep gold particles in ultrapure water or 10mM borate buffer, pH 8.0. If your protein is in PBS then dialyse into borate buffer pH 8.0-8.5 or it does not exceed 5% in volume. Doing conjugation in 10-25mM borate at pH 8.5-9.0 should work for all proteins. Also the BSA for blocking should not be in PBS. Once gold conjugate is blocked effect of sodium chloride is less.

Ahmed Jehanli

Many thanks for your reply. My protein is in 0.1M Glysine, according to the protocol from the vendor of Gold nanoparticles I changed its buffer to 0.5X PBS at pH=7.4 but the protein aggregates in this buffer. I should mention that my GNPs are in 0.01X PBS and when I use 0.01X PBS for protein buffer there is no observation of aggregation with naked eyes but its pH is less than 7.4.

the PI of my protein is 8.5, do not you think that at pH=8.5 in Borate buffer it aggregates?

With borate buffer you mean 0.1M boric acid adjusted with NaOH?

Yes for boric acid plus NaOH

@ Ahmed Jehanli

Thank you. I tried the borate buffer for protein but it aggregated in that condition. I think the problem is the pH because my protein is stored in pH=3 and when I change the buffer to higher pHs it aggregates. Do you have any advice for changing the pH without aggregation of the protein?

Without knowing what the protein is it is difficult to make suggestions. Is it membrane protein, hydrophobic or hydrophilic and what its isoelectric point?