Anti-CD63 antibody [NK1/C3] (ab1318)

Regarding ab81436 (TAPA1).   I am not working on blood or cells. I am working on exosomes. CD81 (TAPA1) is a marker for exosomes, usually I will have exosomes in 100ul of PBS, I would like to ask since your catalog suggested using 100 ul blood and 20ul Ab, so I presume it will be same for exosomes in PBS. but I like to ask should I add 20 ul of this ab onto exosomes pallets alone or 20 ul of this ab into exosomes in 100ul PBS?   and what concentration exactly of ab81200 (PE Control) should I use since I do not have the concentration of ab81436. Would you provide or suggest an exact concentration please.  

ANSWER:

Thank you for contacting us.

If you are not able to easily pellet the exosomes, I would indeed recommend to add 20 µl of the antibody solution to the exosomes in 100 µl PBS. You may need to increase the incubation time and or the volume of ab81436 added, in order to optimise the staining conditions for this particular setting. To our knowledge, this has never been done before, so I am sorry that we cannot give you more detailed information. However, I trust that you will succeed!

Unfortunately, it is NOT possible to determine the EXACT concentration of ab81436: The concentration of the specific TAPA1 antibodies has not been determined before conjugation. Even if it had been, this concentration would be different from the concentration of the antibody after the conjugation procedure, as there is always a loss during the purification steps. Determining the protein concentration after the conjugation does not result in accurate measurements, because Phycoerythrin (PE) is a huge protein itself and thus would give false results, as my colleagues tried to explain earlier (in case you did not receive these emails, see below).

We would therefore suggest to use the PE isotype control ab81200 in the same dilution than ab81436. For example, if you add 20 µl of ab81436 to 100 µl of your sample, than it is recommended to also use 20 µl of the isotype control ab81200 for 100 µl of sample.

Also, I would like to draw your attention to the criteria to record information about the experimental overview and samples (including controls) set out by the flow cytometry community (e.g. http://www.flowcytometryuk.org/). These criteria are known as the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and contain valuable information about isotype controls and FMO controls.

I hope this information is helpful to you. Please do not hesitate to contact me if you need any further advice or information in this regard.

Previously: I can confirm that the concentration of this antibody was not determined. Regrettably, it is not possible to correctly measure the antibody concentration of products with antibodies conjugated to proteins (such as PE). /If there are concentrations given for conjugated antibodies, it is usually the IgG concentration measured before the conjugation took place and therefore only an estimate./ Unfortunately, we do not currently have more information about the concentration of ab81436./ Antibody concentration is usually determined by measuring the protein concentration. This is not useful in this case since PE is also a protein.

Dear Sir/Madam: Recently we purchased ab1318 (anti-CD63) & ab92726 (anti-CD9) antibodies. What is the antibody concentration in these tubes? We don’t need the high accuracy, just the ballpark would be fine.  Thank you,  

ANSWER:

Thank you for contacting us with your question about these antibodies. We don't have exact concentrations for either of these products, but ab1318 is around 100 ug/mL. Since Ab92726 is provided as unpurified tissue culture supernatant, it is not possible to measure the concentration, but supernatant is estimated to have a concentration around 1-3 mg/mL of total protein. I hope this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

Please could you tell me the concentration of antibody or total quantity in the 500 microlitre sized ab1318 ?

Many thanks

ANSWER:

Thank you for your enquiry. The concentration is :100 ug/ml (micrograms per ml). Please do not hesitate to contact us if you need further information.