Dear all, I am doing intracellular staining of one mouse chemokine by indirect labeling method in digested lung cells for flow cytometry study. The secondary antibody is streptavidin Dylight 550, 1;200 dilution used. Compared to the unstained sample, there is a good shift of the fluorescence signal in the stained samples (about 40% positive cells). However, control sample stained with only secondary antibody also shows a similar strong signal. Does anyone have some suggestions on this? The experiment aim is to study which cells express this chemokine (surface makers to identify different cells). Is the current data of any use to answer this question?
Dear Zhenguang
It looks like you have your streptavidin nonspecifically binding to the tissue . You may want to extent the blocking conditions [non-ionic detergents, BSA etc etc]. Please also note that there can be binding of streptavidin to to other targets
Biochem Biophys Res Commun. 1990 Aug 16;170(3):1236-41.
Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin.
Alon R1, Bayer EA, Wilchek M.
Author information
Abstract
Streptavidin binds at low levels and high affinity to cell surfaces, the cause of which can be traced to the occurrence of a sequence containing RYD (Arg-Tyr-Asp) in the protein molecule. This binding is enhanced in the presence of biotin. Cell-bound streptavidin can be displaced by fibronectin, as well as by RGD- and RYD-containing peptides. In addition, streptavidin can displace fibronectin from cell surfaces. The RYD sequence of streptavidin thus mimics RGD (Arg-Gly-Asp), the universal recognition domain present in fibronectin and other adhesion-related molecules. The observed adhesion to cells has no relevance to biotin-binding since the RYD sequence is not part of the biotin-binding site of streptavidin. Since the use of streptavidin in avidin-biotin technology is based on its biotin-binding properties, researchers are hereby warned against its indiscriminate use in histochemical and cytochemical studies.
PMID: 2390089 [PubMed - ind
Ravi is probably right about lack of adequate blocking. What did you use to block?
Also why the secondary step? why not use a direct conjugate, or do it yourself if not available? The Apex kits from life technologies/thermo (or whoever is about to acquire them now) worked really well in the past.
http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/apex-antibody-labeling-kits.html
You could also use their zenon technology to allow you to label a few different primary antibodies, giving you flexibility in the lab.
Thanks a lot. I used anti-CD16/32 to block and do surface staining. Then cells are fixed and permed with the kit from Life Tech, staining with primary antibody and then secondary ab. Currently there is no directly labled conjugate. I may try to buy the antibody labling kits.
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