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Anti myc alexa 647 clone 9b11

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Anti-myc Alexa 647 (clone 9B11) is a fluorescently-labeled antibody that recognizes the myc epitope tag. It is suitable for detection and immunoprecipitation of myc-tagged fusion proteins.

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Lab products found in correlation

Expifectamine, by Thermo Fisher Scientific (4 mentions) Facsaria 2, by BD (4 mentions) Lsr 2 flow cytometer, by BD (4 mentions) Anti flag cy3 clone m2, by Merck Group (3 mentions) Accuri c6, by BD (3 mentions) Fluo 4 am, by Thermo Fisher Scientific (3 mentions) Accuri c6 cytometer, by BD (3 mentions) Accuri c6 flow cytometer, by BD (2 mentions) Facscanto, by BD (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Cxcl12, by Thermo Fisher Scientific (2 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions) Hitrap sp hp cation exchange column, by GE Healthcare (1 mentions) Superdex 75 10 300 gel filtration column, by GE Healthcare (1 mentions) Lsr 2, by BD (1 mentions) Transfection enhancers, by Thermo Fisher Scientific (1 mentions) Fluo 4, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Anti-Myc (254 citations) Anti-Myc (9B11; (26 citations) Alexa 647 (14 citations) Clone 9B11 (9 citations) Anti-myc (clone 9B11, (5 citations)

21 protocols using anti myc alexa 647 clone 9b11

1

Ephrin-B2 Receptor Binding Assay

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Expi293F cells were transfected with 500 ng of pCEP4-myc-EFNB2 (wildtype or mutants) per mL of culture at 2 × 106 cells/mL using Expifectamine (ThermoFisher Scientific). At 24 h post-transfection, the cells were harvested for analysis. A competitor solution was prepared by mixing soluble human IgG1-Fc fused Eph receptor/s (150 nM sEphB2-Fc or 1 μM sEphB3-Fc plus 1μM sEphB4-Fc) and anti-myc Alexa 647 (clone 9B11, 1/250 dilution, Cell Signaling Technology) in PBS-BSA. A non-competitor solution was prepared by diluting anti-myc Alexa 647 (clone 9B11, 1/250 dilution, Cell Signaling Technology) in PBS-BSA. Transfected cells were washed with PBS-BSA and incubated with non-competitor or competitor solution for 5 minutes on ice before the addition of NiV-G-sfGFP-containing medium (1/5 dilution). The mixture was incubated on ice for 30 minutes, washed twice with PBS-BSA, and analyzed on a BD Accuri C6 flow cytometer.
Narayanan K.K., Amaya M., Tsang N., Yin R., Jays A., Broder C.C., Shukla D, & Procko E. (2023). The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins: Selective Ephrin-B2 Decoys for Nipah and Hendra Virus. bioRxiv.
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2

Myc-Epitope Cell Sorting Protocol

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Transfected cells were washed with cold phosphate buffered saline supplemented with 0.2% BSA (PBS-BSA), stained for 40 minutes on ice with excess anti-myc-FITC (rabbit polyclonal, 1/40 dilution, Immunology Consultants Laboratory) or anti-myc-Alexa 647 (clone 9B11, 1/133 dilution, Cell Signaling Technologies), washed twice, and sorted on a BD FACS Aria II at the Roy J. Carver Biotechnology Center. Gating conditions are described in Supplemental Table I. Sorted cells were frozen at −80 °C. To maintain cell viability and mRNA quality during the experiment, samples were sorted for a maximum of 4 hours; to collect greater numbers of cells, libraries were prepared again and frozen sorted cell pellets from multiple days’ experiments were pooled during RNA extraction.
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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3

Screening Library for CCR5-gp120 Interactions

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A human codon-optimized synthetic gene was constructed from gBlocks (IDT) encoding, from N- to C-terminus, a CD5 leader sequence (MPMGSLQPLATLYLLGMLVASVLA) for enhanced expression (24 (link)), HIV-1BaL gp120 a.a. 31–511 (GenBank AAA44191.1: https://www.ncbi.nlm.nih.gov/protein/AAA44191.1) with numbering based on the HXB2 reference strain, a 21-residue gly/ser-rich linker, CD4 domains D1–D2 (a.a. K26–S209), an avi tag, and a FLAG tag. The gene was cloned into pCEP4, and transfected into CXCR4-knockout Expi293F cells. Supernatant was harvested and filtered 4 days post-transfection, and aliquots were stored at −20 °C.
CXCR4-knockout Expi293F cells transfected with a CCR5 SSM library were washed with cold PBS-BSA, and incubated for 60 minutes with culture supernatant from gp120-CD4-expressing cells. Cells were washed twice with PBS-BSA, stained for 20 minutes on ice with chicken anti-DYKDDDDK-FITC (1/133 dilution, Immunology Consultants Laboratory) and anti-myc-Alexa 647(clone 9B11, 1/133 dilution, Cell Signaling Technologies), washed twice, and sorted. Gating conditions for SSM libraries are listed in Supplemental Table I. For the CCR5 combinatorial library that combined mutations predicted to enhance gp120-CD4 binding, the sorted cells were gated for the top 10% of events in the FITC channel, after gating for the top 50% of receptor-positive cells in the Alexa 647 channel.
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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4

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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5

Recombinant CXCL12-sfGFP purification and CXCR4 mutant screening

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A synthetic gene encoding human CXCL12 fused at the C-terminus to superfolder GFP via a 16-residue gly/ser-linker was cloned into pcDNA3.1 (Invitrogen). The plasmid was transfected into CXCR4-knockout Expi293F cells using Expifectamine (Invitrogen) as per the manufacturer’s directions, and culture supernatant was harvested 5 days post-transfection. The supernatant was filtered, and passed over a HiTrap SP HP cation exchange column (GE Life Sciences). The column was washed with 4 CV of 20 mM Tris pH 7.5 / 40 mM NaCl and 2 CV of 20 mM Tris pH 7.5 / 100 mM NaCl, and CXCL12-sfGFP was eluted with 2 CV of 20 mM Tris pH 7.5 / 1.2 M NaCl. The protein was further purified on a Superdex 75 10/300 gel filtration column (GE Life Sciences) with running buffer PBS. The protein was concentrated with a centrifugal filtration device, and protein concentration determined based on sfGFP absorbance at 485 nm. CXCL12-sfGFP was mixed with an equal volume PBS-0.2% BSA, aliquots were flash frozen and stored at −80 °C.
CXCR4-knockout Expi293F cells were transfected with the CXCR4 SSM library. Cells were washed with cold PBS-BSA, and incubated on ice for 40 minutes with excess anti-myc-Alexa 647 (clone 9B11, 1/133 dilution, Cell Signaling Technologies) and 5 μM CXCL12-sfGFP. Cells were washed twice with PBS-BSA, and sorted on a BD FACS Aria II (gating conditions are in Supplemental Table I).
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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6

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.
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. The Journal of Biological Chemistry, 299(10), 105229.
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7

Immunostaining of CXCL12-Expressing Cells

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Transfected X4-KO Expi293F cells were harvested 24 h posttransfection, washed with PBS-BSA, and incubated in 1:200 anti-myc-Alexa 647 (clone 9B11; Cell Signaling Technology) and 10 μM CXCL12-sfGFP for 30 min. Cells were washed and analyzed on a BD LSR II. The preparation of CXCL12-sfGFP is described elsewhere (Heredia et al., 2018).
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. The Journal of Biological Chemistry, 299(10), 105229.
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8

Characterizing CXCL12-sfGFP Binding

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Transfected X4-KO Expi293F cells were harvested 24 h post-transfection, washed with PBS-BSA and incubated in 1:200 anti-myc-Alexa 647 (clone 9B11; Cell Signaling Technology) and 10 μM CXCL12-sfGFP for 30 minutes. Cells were washed and analyzed on a BD LSR II. The preparation of CXCL12-sfGFP is described elsewhere (Heredia et al., 2018 (link)).
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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9

ACE2 expression and RBD binding

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Expi293F cells transfected with pCEP4- myc-ACE2 plasmids were collected 24 h post-transfection (600 × g, 60 s), washed with ice-cold Dulbecco’s phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS-BSA. After 30 minutes incubation on ice, cells were washed twice in PBS-BSA and analyzed on a BD Accuri C6 using instrument software. Gates were drawn around the main population based on forward and side scatter properties, followed by a gate for medium Alexa 647 fluorescence to control for any expression differences between mutants. Mean superfolder GFP (sfGFP) fluorescence was measured and background fluorescence of negative cells was subtracted. All replicates were from independent, distinct samples.
Chan M.C., Chan K.K., Procko E, & Shukla D. (2021). Machine learning guided design of high affinity ACE2 decoys for SARS-CoV-2 neutralization. bioRxiv.
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10

ACE2-RBD Binding Assay by Flow Cytometry

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The flow cytometry analysis of ACE2-S Binding were performed as described previously [17] (link). Briefly, 293 T cells were transfected with pCEP4-MYC-ACE2 or pcDNA3-SARS-CoV-2-S-RBD-sfGFP plasmids (500 ng DNA per mL of culture at 2 × 106 / mL) using lipofectamine 2000 (ThermoFisher). The medium with indicated RBD-sfGFP were collected from 293 T cells which transfected with pcDNA3-SARS-CoV-2-S-RBD-sfGFP plasmid after 48 h. After pretreated by indicated drugs for 24 h, 293 T cells which transfected with pCEP4-MYC-ACE2 plasmid were washed with ice-cold PBS-BSA, and incubated for 30 min on ice with medium containing RBD-sfGFP and the anti-MYC Alexa 647 (clone 9B11, Cell Signaling Technology). Then, cells were washed twice with PBS-BSA and analyzed by the flow cytometer BD FACSCanto (Becton Dickinson).
Tsai M.S., Shih W.T., Yang Y.H., Lin Y.S., Chang G.H., Hsu C.M., Yeh R.A., Shu L.H., Cheng Y.C., Liu H.T., Wu Y.H., Wu Y.H., Shen R.C, & Wu C.Y. (2022). Potential inhibitor for blocking binding between ACE2 and SARS-CoV-2 spike protein with mutations. Biomedicine & Pharmacotherapy, 149, 112802.
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