Goat anti-Mouse IgG H&L (20nm Gold) secondary antibody (ab27242)

Re: Problems with Gold-conjugated secondary antibodies ab27241 and ab27242 in EM with neuroblastoma cells.

Hi, thanks for getting back to me.

The cells I use are neuroblastoma cells with rat origin. I transfect my cells with the GFP mutant synaptopHluorin, this enables me to image the hydrated cells by fluorescence microscopy.

I then fix my cells with PFA and permeabilised with Triton (I have tried concentrations between 0.1- 1%)

I then labelwith anti GFP primary antibody andgold conjugated secondary antibody to allow detection of the labelled areas in the hydrated cells by atmospheric SEM.

Generally the results show single particles of gold scattered throughout the cell. Even in samples where I have attempted to label actin filaments of endogenous proteins I do not see localisation to specific cellular patterns. It has also been questioned that surely more gold particles should be observed, taking into account the abundance of cellular structures labelled by the primary antibody. (Fluorescence assay have been carried out to determine the specificity of the primary antibody and have shown perfect colocalisation.)

I have seen better results when I permeabilse with 1% Triton, although here I used a new transfection agent, so far I have not been able to repeat these results.

I think a problem may be in either the specificity of the gold conjugated secondary antibody or the penetration of the gold particles into the hydrate cells. To solve this I can see two options 1) use smaller gold particles. This would be ok in normal SEMs although the resolution of my current instrument for ASEM is ˜20nm, so I'm not too keen to use much smaller particles.

2) Alternative methods in fixation/permeabilisation to allow easier passage.

I would be very grateful for your thoughts on my problem.

Thanks,

ANSWER:

Thank you very much for sending all these details which enable us to investigate the problem.

Thank you also for your patience while I was inquiring with the laboratory.

Firstly I can confirm that we have not had any otherreports of any problems with these towbatches ofantibodies mentioned.

In regards to solve the problem, I would like firstly to ask you whether you have checked whether the primary antibody is working. Have you used this primary antibody already on similar fixed slides and obtained a good signal?

In regards to two secondary antibodies, the laboratory recommends toreturnto using 1% Triton to rule out the change of transfection agent as being the cause of the issues.

We can also recommend(on advice of the laboratory)to try using a smaller gold particle (as for exampleab27244) and silver enhance this to obtain a larger signal which should be visible under the ASEM that they are using. The product needed to carry out this silver enhancing step is commercially available. If you would like us to source it for you, please let me know and I see what I can do. The silver enhancing step consists of simply adding an equal volume of the supplied silver enhancer and initiator to the sample after incubation with the secondary antibody (the conjugate being used) has been carried out. The silver particles will then attach to the gold particles which are present in the sample, growing them to a larger size and therefore making them large enough to be visible under ASEM.

In case that the suggestions will not improve the results, and the primary antibody is excluded as possible source of problem, we will be happy to replace or refund these antibodies for you in accordance with our Abpromise policy.

Please do let me know if you have any questions or concerns.

I am looking forward to hear back from you to know whether the problem has been resolved. Thank you for your cooperation.

Hi, We have a customer who is interested in purchasing this product however firstly they have asked me to contact you to double check that you do not have any further references for this product? Thank you for your time and I look forward to your reply. Kind regards,

ANSWER:

Thank you for your inquiry.

Unfortunately, we do not currently have more information about this product. We will update the on-line datasheet for this product as soon as we receive additional data.

We apologize for any inconvenience this may cause.