Anti flag cy3 clone m2

Manufactured by Merck Group

Anti-Flag Cy3 (clone M2) is a laboratory reagent produced by Merck Group. It is a fluorescently labeled antibody that specifically binds to the FLAG epitope, a commonly used protein tag. The Cy3 fluorescent dye is conjugated to the antibody, allowing for detection and visualization of FLAG-tagged proteins.

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Lab products found in correlation

Anti myc alexa 647 clone 9b11, by Cell Signaling Technology (3 mentions) Facsaria 2, by BD (2 mentions) Lsr 2 flow cytometer, by BD (1 mentions)

Spelling variants of this product

Anti-FLAG (2121 citations) Anti-FLAG M2 (1022 citations) FLAG M2 (383 citations) Anti-FLAG (clone M2, (69 citations) FLAG (clone M2, (25 citations) Clone M2 (23 citations) Anti-FLAG M2-Cy3 (5 citations) Anti-FLAG-Cy3 (3 citations)

3 protocols using anti flag cy3 clone m2

Fluorescence-Activated Cell Sorting for Protein-Protein Interactions

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Cells transfected with the libraries were washed with cold PBS-BSA and incubated with 1/300 anti-myc Alexa 647 (clone 9B11; Cell Signaling Technology) plus 1/300 anti-Flag Cy3 (clone M2; Sigma-Aldrich) in PBS-BSA for 30 minutes before being washed twice and resuspended in cold PBS-BSA supplemented with 1/100 fetal bovine serum (FBS). Cells were sorted on a BD FACS Aria II at the Roy J. Carver Biotechnology Center. Cells were first gated by forward-side scatter and side scatter for the main population of cells. For sorting based on surface expression, cells positive for the myc tag (Alexa 647 fluorescence) were collected. For sorting based on high BiFC signal, within the expression gate the top 5% of cells for YFP/Venus fluorescence relative to surface receptor expression were collected. Cells were sorted for no longer than 4 h to maintain high viability and were collected in tubes coated overnight in FBS. Collected cells were centrifuged and the pellets stored at −80 °C.

Gill K.S., Mehta K., Heredia J.D., Krishnamurthy V.V., Zhang K, & Procko E. (2023). Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis. bioRxiv.

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Fluorescence-Activated Cell Sorting Protocol

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Cells transfected with the libraries were washed with cold PBS-BSA and incubated with 1/300 anti-myc Alexa 647 (clone 9B11; Cell Signaling Technology) plus 1/300 anti-Flag Cy3 (clone M2; Sigma-Aldrich) in PBS-BSA for 30 min before being washed twice and resuspended in cold PBS-BSA supplemented with 1/100 fetal bovine serum. Cells were sorted on a BD FACS Aria II at the Roy J. Carver Biotechnology Center. Cells were first gated by forward-side scatter and side scatter for the main population of cells. For sorting based on surface expression, cells positive for the myc tag (Alexa 647 fluorescence) were collected. For sorting based on high-BiFC signal, within the expression gate, the top 5% of cells for YFP/Venus fluorescence relative to surface receptor expression were collected. Cells were sorted for no longer than 4 h to maintain high viability and were collected in tubes coated overnight in fetal bovine serum. Collected cells were centrifuged and the pellets stored at −80 °C.

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Bimolecular Fluorescence Complementation Assay

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As outlined above, CCR5 and CXCR4 were cloned into pCEP4 with N-terminal signal peptides from influenza hemagglutinin, followed by a c-myc or FLAG epitope tag, a 6-residue spacer, and receptor residues 2–352. Human mGluR3 was cloned into pCEP4 with the signal peptide of HLA class I A-2 α chain, a c-myc or FLAG epitope tag, and a 3-residue linker to receptor residues R30-L879. At the cytosolic C-termini of the receptors, immediately prior to the stop codons, were fused N- (VN: a.a. V1-A154) or C-terminal (VC: a.a. D155–K238) residues of Venus (mutant I152L), a yellow fluorescent protein variant with improved signal-to-noise for the detection of protein interactions by BiFC (27 (link), 28 (link)). Pairs of VN and VC fused receptors (300 ng of each plasmid per ml of 2 × 10⁶ cells) were co-transfected into CXCR4-knockout Expi293F cells using Expifectamine, and cells were analyzed by flow cytometry 22–24 hours post-transfection. Washed cells were stained with anti-FLAG-Cy3 (clone M2, 1/200 dilution, Sigma) and anti-myc-Alexa 647 (clone 9B11, 1/200 dilution, Cell Signaling Technologies), washed twice, and analyzed on a BD LSR II flow cytometer with appropriate 3-color compensation.

Heredia J.D., Park J., Brubaker R.J., Szymanski S.K., Gill K.S, & Procko E. (2018). Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning. Journal of immunology (Baltimore, Md. : 1950), 200(11), 3825-3839.

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