Biotin Anti-TLR9 antibody [5G5] (ab17236)

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Unfortunately, all the references and images available are already on the datasheet. We are continuously updating our datasheets, and as soon as we gather further characterisation details about it, we will share it on the Website.

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I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your e-mail regarding ab17236 - Mouse monoclonal [5G5] to TLR9 (Biotin). We are very sorry to hear the there are still problems with this product and have been discussing this complaint with some colleagues. We looked very carefully the information you provided to us. You mentioned that you tried staining 1x10E6 cells with between 0.1 and 0.5 ug/ml of primary antibody. What is the volume you actually used? We would advise you to use a concentration of 10 ug/ml ( 2 ug/200 ul). It seems that concentration of 0.1 ug/ml are very low. We use the following fixation and permeabilization protocol: - Wash the cells 2x with cold PBS. Temperature control at this point is of vital importance. Note: Cells should be kept on ice any time they are not spinning, and cells should be spun in a temperature controlled centrifuge at 4°C. - Fix the cells with 1% paraformaldehyde for 15 minutes at 4°C. Note: it is crucial to make sure that the cells are resuspended well. - Wash the cells 2x with ice cold PBS. - Permeabilize the cells with 100% ice cold methanol added drop wise while the cells are vortexing. It is crucial to make sure that the cells are resuspended thoroughly. Leave in methanol for 15 minutes. - Wash the cells 2x with cold PBS - Add approximately 1x10E6 cells to each FACS tube and wash with 1 ml of 0.1% saponin diluted in PBS with 2% fetal bovine serum added. - Resuspend cells in 1 ml of 0.1% saponin + 2% FBS and allow to sit for 30 minutes blocking at RT. - Add primary antibody We are not sure why you washed the cells between the permeabilization step and the primary antibody incubation. We would like to advise you to follow the above protocol (if this is not the protocol you normally use). I hope this information will be useful for you, please do not hesitate to contact Abcam if you still have further questions.

Thank you for your enquiry. This Ab is cited int following paper being used for WB: Ahmad-Nejad, P et al; Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol 2002, 32: 1958 Contained within this paper is a Western blot image pertaining to this product. If you need any further assistance then please get back in touch with us.