For co-immunoprecipitation, Monoclonal ANTI-FLAG M2 Antibody from Sigma (F3165 RRID:AB_259529 ) and anti-COPS7b antibody from Abcam (ab124718 RRID:AB_10971678 ) were used at 1:50 dilution. For detection in co-immunoprecipitation experiments, primary Monoclonal ANTI-FLAG M2 Antibody from Sigma was diluted at 1:1000, primary anti-COPS7b antibody from Abcam was used at 1:1000. For detection of protein levels in an ‘’around-the-clock’’ experiment primary anti-COPS7b antibody from Abcam was used at 1:1000 and Anti-βIII Tubulin mAB from Promega (G7121 RRID:AB_430874 ) was used at 1:500 dilutions. The probing of the secondary antibody was done at 1:10000 for IRDye 680–goat anti-mouse IgG (926–32220 RRID:AB_621840 ; Licor, Lincoln, NE) and 1:10000 for IRDye 800–goat anti-rabbit IgG (926–33210 RRID: AB_10796098 ; Licor).
Anti flag m2 monoclonal antibody
Manufactured by Merck Group
Sourced in United States, Germany, Japan, United Kingdom
The Anti-FLAG M2 monoclonal antibody is a laboratory reagent used for the detection and purification of proteins tagged with the FLAG peptide sequence. It is a mouse monoclonal antibody that binds specifically to the FLAG peptide with high affinity. The antibody is commonly used in various techniques, such as immunoprecipitation, Western blotting, and immunoaffinity chromatography, to identify and isolate FLAG-tagged proteins from cell lysates or other biological samples.
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Lab products found in correlation
Flag peptide, by Merck Group (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Clarity western ecl substrate, by Bio-Rad (6 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (4 mentions)
Laemmli sample buffer, by Bio-Rad (4 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
Anti flag agarose beads, by Merck Group (4 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (4 mentions)
Immobilon p membrane, by Merck Group (4 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Goat anti rabbit igg dylight 680 conjugate, by Thermo Fisher Scientific (4 mentions)
Goat anti mouse igg dylight 800 conjugate, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Anti flag m2 magnetic bead, by Merck Group (3 mentions)
Protein g dynabead, by Thermo Fisher Scientific (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
293 protocols using anti flag m2 monoclonal antibody
1
Co-immunoprecipitation Antibody Optimization
2
Immunofluorescence Staining of Transfected COS-7 Cells
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Forty-eight hours after transfection, COS-7 cells transfected with pcDNA3β-FLAG constructs were fixed with 3.7% formaldehyde in PBS and examined by immunofluorescence staining with anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), followed by secondary Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA). Stained cells were mounted in VECTASHIELD mounting medium and observed using a BX50 fluorescence microscope (Olympus, Tokyo, Japan). Cytospins of murine bone-marrow cells transduced with pMY-IG/IRES-GFP viral constructs encoding FLAG-CALM-AF10, FLAG-CALMNES4A-AF10, FLAG-NES2-AF10 or FLAG-mAF10 were fixed with 4% paraformaldehyde and stained with anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) and anti-KMT4/DOT1L polyclonal rabbit antibody (Abcam, Cambridge, MA, USA), followed by secondary Alexa Fluor 568-conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen), respectively. Stained bone-marrow cells were mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) or Prolong Gold (Invitrogen) and observed under a BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan) or a FluoView FV10i confocal laser scanning microscopy (Olympus).
3
Protein expression and western blotting analysis
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Stable overexpression cells were grown up to 90% confluency in 6 well plates and lysed in RIPA buffer with 1 mM DTT, 1 mM benzamidine, 1 mM PMSF and 1× protease inhibitor cocktail. Cell resuspensions were incubated for 15 min on ice, followed by short sonication. The supernatants of lysates were collected after centrifugation at 10,000 rpm for 10 min at 4 °C. The samples were heated with 1× SDS loading buffer at 98 °C for 5 to 10 min. Following 10% or 15% SDS-PAGE and wet blot transfer, the blots were probed with one of the following primary antibodies in TBST: monoclonal anti-FLAG® M2 antibody (Merck KGaA, Cat. No. F1804, Darmstadt, Germany), mTOR (phosphor-S2448) polyclonal antibody (Bioworld, Cat. No. BS4706, Nanjing, China), mTOR (S2442) polyclonal antibody (Bioworld, Cat. No. BS3611, Nanjing, China), anti-HIF-1α (Sangon, Cat. No. D162108, Shanghai, China); anti-LC3 (abcam, Cat. No. ab51520, Cambridge, UK); anti-p62 (abcam, Cat. No. ab56416, Cambridge, UK); anti-GFP (Proteintech, Cat. No. 66002-1-Ig, Wuhan, China), anti-GAPDH (Proteintech, Cat. No.60004-1-Ig, Wuhan, China).
4
Western Blotting of NFATC1, NFATC2, and DYRK1A
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Western blotting was performed as previously described.31 (link) Monoclonal anti‐flag M2 antibody (F1804; Merck KGaA) was used to detect exogenous NFATC1. NFATC1 monoclonal antibody (MA3‐024, Thermo Fisher Scientific, Inc.) and NFATC2 monoclonal antibody (MA1‐025, Thermo Fisher Scientific, Inc.) were used to detect NFATC1 and NFATC2, respectively. DYRK1A polyclonal antibody (2771, Cell Signaling Technology, Inc.) was used to detect DYRK1A, and an anti‐β‐actin monoclonal antibody (A1978, Merck KGaA) was used to detect ACTB as a loading control. IRDye 680 goat anti‐rabbit IgG (c10207‐01, Li‐COR, Lincoln) and IRDye 800CW goat anti‐mouse IgG (C11026‐03, Li‐COR, Lincoln) were used as secondary antibodies. Detection and quantification were performed using the Li‐COR Odyssey imaging system.
5
Protein Extraction and Analysis from Seeds
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Proteins were extracted from frozen and homogenized seeds. For 4 mg seed material, 100 µL freshly prepared extraction buffer (4% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 2 mM phenylmethane sulfonyl fluoride, 0.1 M Tris pH 8.5) was added. Samples were immediately, vigorously vortexed for at least 2 min. Afterwards, the samples were incubated at 80 °C for 3 min and centrifuged (10 min, 20,810 g, room temperature). The supernatant was transferred to a new tube and was mixed with 4 × Läemmli buffer. For SDS-PAGE and western blot analysis, 10 µL of with 4 × Läemmli buffer diluted protein extract was loaded on an SDS gel. For western blot analysis, proteins were detected using an anti-GFP antibody (diluted 1:5,000, BioLegend), monoclonal anti-c-MYC antibody (1:5000, Sigma) and monoclonal anti-FLAG M2 antibody (1:5,000, Merck) followed by the anti-Mouse IgG (whole molecule)-alkaline phosphatase (diluted 1:30,000, Merck). The SDS gel serving as loading control was stained with coomassie.
6
Antibody Detection and Purification
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Monoclonal ANTI-FLAG M2-Peroxidase (HRP) antibody produced in mouse (Merck, Rahway, NJ, USA), monoclonal ANTI-FLAG M2 antibody produced in mouse (Merck), mouse anti Strep-Tag Classic antibody, clone Strep-Tag II purified (BioRad), mouse anti Strep-Tag Classic antibody, clone Strep-Tag II HRP conjugated (BioRad), and goat polyclonal anti-mouse IgG (H&L) Antibody DyLight 649 Conjugated Pre-Adsorbed (Rockland Immunochemicals) were used.
7
Protein Extraction and Western Blot Analysis
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Proteins were extracted from frozen and homogenized seeds. To 4 mg seed material, 100 µL freshly prepared extraction buffer (4 % (w/v) SDS, 2 % (v/v) β-mercaptoethanol, 2 mM phenylmethane sulfonyl uoride, 0.1 M Tris pH 8.5) were added. Samples were immediately vigorously vortexed for at least 2 min.
Afterwards, the samples were incubated at 80 °C for 3 min and centrifuged (10 min, 20810 g, room temperature). The supernatant was transferred to a new tube and was mixed with 4x Lämmli buffer. For SDS-PAGE and western blot analysis, 10 µL of with 4x Lämmli buffer diluted protein extract was loaded on a SDS gel. For western blot analysis, proteins were detected using an anti-GFP epitope tag antibody (diluted 1:5,000, BioLegend), monoclonal anti-c-MYC antibody (1:5,000, Sigma) and monoclonal anti-FLAG M2 antibody (1:5,000, Merck) followed by the anti-Mouse IgG (whole molecule)-Alkaline Phosphatase (diluted 1:30,000, Merck). The SDS gel serving as loading control was stained with coomassie.
Afterwards, the samples were incubated at 80 °C for 3 min and centrifuged (10 min, 20810 g, room temperature). The supernatant was transferred to a new tube and was mixed with 4x Lämmli buffer. For SDS-PAGE and western blot analysis, 10 µL of with 4x Lämmli buffer diluted protein extract was loaded on a SDS gel. For western blot analysis, proteins were detected using an anti-GFP epitope tag antibody (diluted 1:5,000, BioLegend), monoclonal anti-c-MYC antibody (1:5,000, Sigma) and monoclonal anti-FLAG M2 antibody (1:5,000, Merck) followed by the anti-Mouse IgG (whole molecule)-Alkaline Phosphatase (diluted 1:30,000, Merck). The SDS gel serving as loading control was stained with coomassie.
8
STAT5 Transcription Factor Assay
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TransAM® STAT5 transcription factor assay kit was purchased from Active Motif (Carlsbad, CA). TurboFect Transfection Reagent was purchased from Thermo Scientific (Hudson, NH, USA). Dual-luciferase reporter assay system, pGL-STAT5 and pRL-TK vector were obtained from Promega Corporation (Promega, Madison, WI, USA). The plasmid encoding with STAT5B gene (pCMV6-AC-GFP-STAT5B) and anti-GFP antibody were purchased from OriGene Technologies (Rockville, MD, USA). Prolactin, ANTI-FLAG® M2 Magnetic Beads, Monoclonal ANTI-FLAG® M2 antibody and MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Quantitative ELISA for Aβ Detection
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Sandwich ELISAs used for Aβ detection were performed as previously described [39 (link)]. Briefly, Aβ and Aβ adducts with substrate TMD in conditioned media was captured with Ab5 antibody (human Aβ1–16 specific) and detected with horseradish peroxidase mAb 4G8 (Covance, Princeton, NJ, USA). Synthetic Aβ1–40 was used as standard. All ELISAs were developed with TMB substrate (KPL, Gaithersburg, Maryland, USA). Bis-Tris precast gels (Biorad, Hercules, CA, USA) were used for all SDS-PAGE. Monoclonal anti-FLAG M2 antibody (Sigma) and Aβ1–16 antibody 6E10 (Covance) were used for Western blotting. Antisera against SPPL2a, SPPL2b, hSPP and pSPP were generated as previously described [42 (link)] and were used at dilution of 1:1000 for Western blotting. Anti-CD74 In-1 antibody (BD Bioscience, San Jose, CA, USA) was used for A20 cell lysate Western blotting at dilution of 1:1000.
10
Antibody Characterization Protocol
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