96-well plates were seeded with 100,000 of MCA-N4 tumor cells or parental MCA205 cells as control 4–5 h before the start of the experiment. T cells isolated from tumors (see above) were allowed to rest in prewarmed complete RPMI at 37°C for 1 h. 100,000 T cells were added to each well and incubated at 37°C for 20 min. Ice-cold 4% PFA was then added for a final concentration of 1.6% PFA, and cells were fixed on ice for 10 min. Cells were then spun down and permeabilized with ice-cold 90% MeOH for 20 min on ice. Plates were spun down and washed with FACS buffer (PBS with 3% FBS) and stained for 30 min at room temperature with anti-phospho-p44/42 MAPK (Cell Signaling Technology; clone E10), BV650-conjugated anti-CD8α (BioLegend; clone 53–6.7), and FITC-conjugated anti-Thy1.1 (eBioscience; clone HIS51). Primary antibodies were then washed off before the addition of Alexa Fluor 647–conjugated goat anti-mouse IgG1 (Invitrogen). Cells were incubated for 30 min at room temperature before being washed and analyzed by flow cytometry.
Alexafluor647 conjugated goat anti mouse igg1
Manufactured by Thermo Fisher Scientific
Sourced in Panama
AlexaFluor647-conjugated goat anti-mouse IgG1 is a secondary antibody that specifically binds to the IgG1 subclass of mouse immunoglobulins. The antibody is labeled with the AlexaFluor647 fluorescent dye, which can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Dylight488 donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Mouse anti pannav igg1, by Merck Group (2 mentions)
Alexafluor555 conjugated goat anti mouse igg2a, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Anti phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti mouse or anti rabbit igg, by GE Healthcare (1 mentions)
Cdc42, by BD (1 mentions)
4 protocols using alexafluor647 conjugated goat anti mouse igg1
1
T Cell Activation and Signaling Assay
2
Generating NT2-AnkG-GFP Expression Construct
3
Generating NT2-AnkG-GFP Expression Construct
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To generate an NT2-AnkG-GFP expression construct, we cloned Ank3-exon1e cDNA (Open Biosystems, catalogue #MMM1013-64925) into pEGFP-N1. Vann Bennett provided a rat NT3-AnkG270-GFP plasmid and affinity-purified rabbit anti-All AnkG antibodies (raised against a large C-terminus fusion protein). Isoform-specific rabbit antibodies against portions of the NT1-AnkG, NT2-AnkG and NT3-AnkG peptides were raised (YenZym, Inc., S. San Francisco, CA) and affinity-purified using methods described previously(22 (link)). Antigenic peptides used were derived from the human sequences, with a C-terminal cysteine residue added for conjugation: NT1 (NT1-AnkG residues 8–29): SPAGTEDSAPAQGGFGSDYSRSSRKSC; NT2 (residues 2–21): SEEPKEKPAKPAHRKRKGKKC; NT3 (residues 2–21): AHAASQLKKNRDLEINAEEEC. Purchased primary antibodies were mouse N-terminal isoform non-selective, called here “All AnkG” (IgG2a, clone N106/36; UC Davis/NIH NeuroMab Facility; Davis, CA), mouse anti-PV (IgG1, clone Parv-19, Sigma-Aldrich; St. Louis, MO), and mouse anti-PanNaV (IgG1, Sigma-Aldrich). Corresponding secondary antibodies were dylight488 donkey-anti-rabbit IgG (Jackson ImmunoResearch; West Grove, PA), AlexaFluor647-conjugated goat anti-mouse IgG1 (Invitrogen; Eugene, OR), AlexaFluor555-conjugated goat anti-mouse IgG2a (Invitrogen), and, for western blots, HRP-conjugated anti-rabbit IgG (Jackson).
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Immunoblotting and Immunoprecipitation Protocols
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The primary antibodies used for immunoblotting and immunoprecipitations: p120 (pp120), E-cadherin (34-E-cadherin), β-catenin, phospho-p120 (pY228), phosphotyrosine (pY20), Rac1, and Cdc42 were from BD Bioscience (San Jose, CA). β-actin (C-2), CD148 (143-41), and RhoA (119) were from Santa Cruz Biotechnology (Santa Cruz, CA). Src was from Upstate Biotechnology (Lake Placid, NY). Phospho-Src (pY529) was from Invitrogen Corporation (Carlsbad, CA). Secondary antibodies for immunoblotting: HRP-conjugated anti-mouse or anti-rabbit IgG were from GE Healthcare Bio-Sciences (Pittsburgh, PA). For immunoprecipitation of E-cadherin, anti-E-cadherin (HECD-1) from Takara Bio Company (Madison, WI) was used. For flow cytometry, phycoerythrin (PE)-conjugated anti-CD148 (143-41) from R&D Systems (Minneapolis, MN) was used. Immunofluorescence staining was performed using anti-E-cadherin (HECD-1, Takara Bio), anti-p120 (pp120, BD Bioscience), and the following secondary antibodies; FITC-conjugated anti-CD148 (143-41, Santa Cruz Biotechnology), Alexa Fluor 546-conjugated goat anti-mouse IgG2a (Invitrogen Corporation, Carlsbad, CA), and Alexa Fluor 647-conjugated goat anti-mouse IgG1 (Invitrogen Corporation).
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