Anti-Collagen II antibody [2B1.5] (Biotin) (ab79127)

HI,

This is with regard to anti collagen II antibody (2B1.5), it is a mouse monoclonal antibody to collagen II conjugated to biotin,

I would like to know some details with regard to the usage protocol of this antibody for flow cytometry application.

·        

How are the cells with ECM processed for flow cytometry if they are cultured in vitro ? Does it have any effect on the specificity of detection once digested with enzymes? The concentration of enzymes used for digestion to be

suggested.

·        

What are the positive and negative controls tobe used ?

·        

Could you suggest the isotype and the secondary antibody used for flow cytometry? 

I would appreciate the information you would provide to use this antibody for detection of COL2A  with regard to the usage and any other usage related information .

Thank you.

ANSWER:

Thank you very much for your inquiry.

I have found meanwhile the following references where flow cytometry for collagen proteins has been described:

http://ukpmc.ac.uk/articles/PMC2748431

http://www.pnas.org/content/106/42/17892.full

http://www.sciencedirect.com/science/article/pii/S1063458400903522

http://scholarlyrepository.miami.edu/dissertations/2559/

In these publications the intracellular collagen content is measured. I am unfortunately not aware of any method quantifying extracellular matrix by flow cytometry. To measure then the intracellular collagen content, depending on the cells, they were prepared by detaching them with EDTA 1.5mM. As you will differentiate (with a negative control) the amount of background due to protein which is extracellular to the one which is intracellular, I do not think that the digestion method is very crucial. I am however not a specialist in this domain and suggest you to do further research in this domain.

Positive controls to be used are cells expressing the protein as well as an antibody with fluorochrome giving a signal in the desired color. Cells expressing collagen II are for example chondrocytes, hepatocarcinoma cells or COLO205 cell. Many others cells surely express collagen II as well.

Negative controls should include "unstained cells", "isotype stained" and "secondary or avidin only". If you are doing the intracellular staining, I strongly recommend to perform those controls, as well as the positive controls and staining, also on unpermeablized cells. This will allow you to determine exactly the staining and the background if there is some.

For the ab79127 I would recommend to use fluorochrome coupled avidin as this antibody is already biotinylated. Unfortunately we do not have avidin in our catalogue for the moment. However on www.biocompare.com and many other companies you will surely be able to find a suitable avidin or streptavidin.

As an isotype control I would recommend the ab18454, as it is also an mouse IgG2a, kappa and biotinylated.

I hope this information is helpful for you. Please do not hesitate to get back to us should you have any additional question or concern.

I wish you good success for your research.