Rat anti ha clone 3f10

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Rat anti-HA clone 3F10 is a laboratory reagent used for the detection and purification of proteins tagged with the HA (Hemagglutinin) epitope. It is a monoclonal antibody derived from rat and specifically binds to the HA tag.

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Anti-HA (475 citations) Rat anti-HA (41 citations) Rat anti-HA (3F10, (9 citations) HA (3F10; (5 citations) Anti-HA (3F10), (4 citations) Clone 3F10 (3 citations) Anti-HA (clone 3F10, (2 citations)

6 protocols using rat anti ha clone 3f10

Gametocyte Immunofluorescence Assay Protocol

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Gametocyte immunofluorescence assays were performed as previously described (58 (link)). For HA and α-tubulin staining, purified cells were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde in PBS for 1 hour, permeabilized with 0.1% Triton X-100/PBS for 10 min, and blocked with 2% BSA/PBS for 2 hours. Primary antibodies were diluted in blocking solution (rat anti-HA clone, 3F10, 1:1000; mouse anti–α-tubulin clone, DM1A, 1:1000; both from Sigma-Aldrich). Anti-rat Alexa 594, anti-mouse Alexa 488, anti-rabbit Alexa 488, and anti-rabbit Alexa 594 were used as secondary antibodies together with DAPI (4′,6-diamidino-2-phenylindole) (all from Life Technologies), all diluted at 1:1000 in blocking solution. Confocal images were acquired with a LSM700 or a LSM800 scanning confocal microscope (Zeiss).

Balestra A.C., Koussis K., Klages N., Howell S.A., Flynn H.R., Bantscheff M., Pasquarello C., Perrin A.J., Brusini L., Arboit P., Sanz O., Castaño L.P., Withers-Martinez C., Hainard A., Ghidelli-Disse S., Snijders A.P., Baker D.A., Blackman M.J, & Brochet M. (2021). Ca2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG. Science Advances, 7(13), eabe5396.

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Antibodies for Western Blot and IP

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Primary antibodies used in Western blot and IP experiments were rat anti-HA clone 3F10 (Sigma-Aldrich), mouse anti-Flag clone M2 (Sigma-Aldrich), mouse anti-V5 Tag (Thermo Fisher Scientific), mouse anti-TRIM8 (Santa Cruz Biotechnology, sc-398878), mouse anti-IRF7 clone G-8 (Santa Cruz Biotechnology, sc-74472), rabbit anti-pIRF7 (Ser⁴⁷⁷) clone D7E1W (no. 12390, Cell Signaling), rabbit anti-ubiquitin (linkage-specific K63) clone EPR8590-448 (Abcam), rabbit anti-Pin1 (no. 3722, Cell Signaling), and mouse anti–β-actin (Sigma-Aldrich). Secondary antibodies used were anti-mouse, anti-rat, and anti-rabbit horseradish peroxidase (HRP)–conjugated (GE Healthcare Life Sciences).
For immunofluorescence and flow cytometry, we used Alexa Fluor 488 mouse anti-IRF7 (pS477/pS479) clone K47-671 (BD Biosciences), mouse anti–IFN-α phycoerythrin (PE) clone LT27:295 (Miltenyi Biotec), mouse allophycocyanin (APC) anti–BDCA-4 clone REA380 (Miltenyi Biotec), mouse fluorescein isothiocyanate (FITC) anti-CD123 clone AC145 (Miltenyi Biotec), and rabbit anti-TRIM8 (Thermo Fisher Scientific) antibodies. Secondary antibody (for TRIM8 labeling) was Alexa Fluor 546 goat anti-rabbit (Thermo Fisher Scientific).

Maarifi G., Smith N., Maillet S., Moncorgé O., Chamontin C., Edouard J., Sohm F., Blanchet F.P., Herbeuval J.P., Lutfalla G., Levraud J.P., Arhel N.J, & Nisole S. (2019). TRIM8 is required for virus-induced IFN response in human plasmacytoid dendritic cells. Science Advances, 5(11), eaax3511.

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Immunofluorescence Assay for HA Staining

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Immunofluorescence assays were performed as described in ref. ^{56 (link)}. Briefly, for HA staining, purified cells were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde in PBS for 1 h, permeabilised with 0.1% Triton X-100/PBS for 10 min and blocked with 2% BSA/PBS for 2 h. Primary antibodies were diluted in blocking solution (rat anti-HA clone 3F10, 1:1000 from Sigma-Aldrich, reference 000000011867431001). Anti-rat Alexa488 (Life Technologies, reference A-11006) was used as a secondary antibody together with 4′,6-diamidino-2-phenylindole (DAPI) and diluted 1:1000 in blocking solution. Confocal images were acquired with a LSM800 scanning confocal microscope (Zeiss).

Fang H., Gomes A.R., Klages N., Pino P., Maco B., Walker E.M., Zenonos Z.A., Angrisano F., Baum J., Doerig C., Baker D.A., Billker O, & Brochet M. (2018). Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites. Nature Communications, 9, 4248.

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Investigating EDEM3 and Protein Trafficking

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HEK293T and HeLa cells were from the European Collection of Animal Cell Cultures. Rabbit anti-EDEM3 (E8906) and rat anti-HA, clone 3F10 (cat: 12158167001) were from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-HA.11, clone 16B12 (cat: 901503) was from BioLegend (San Diego, CA, USA). Rabbit anti-α1-anti-trypsin (A0012) was from Dako (Jena, Germany). Mouse anti-tyrosinase (T311) antibody (sc-20035) and mouse anti-MAN1B1 antibody (sc-393145) was from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit polyclonal antibodies were generated in house for tyrosinase [63 ] and EDEM3 [64 ]. Rabbit anti-calnexin (ab22595), rat anti-tubulin (ab6161) and mouse anti-actin (ab8224) antibodies were from Abcam (Cambridge, UK). Endoglycosidase H (EndoH, P0702S) and Peptide-N-Glycosidase F (PNG-ase F, P0704S) were from New England Bioland (Ipswich, MA, USA). Kifunensine (sc-201364), MG132 (sc-201260) and all other chemicals were from Santa Cruz Biotechnology unless specified.

Manica G., Ghenea S., Munteanu C.V., Martin E.C., Butnaru C., Surleac M., Chiritoiu G.N., Alexandru P.R., Petrescu A.J, & Petrescu S.M. (2021). EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning. International Journal of Molecular Sciences, 22(4), 2172.

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Gametocytes Immunofluorescence Assay

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Gametocytes immunofluorescence assays were performed as previously described (Volkmann et al., 2012 (link)). For HA, c-myc and α-tubulin staining, purified cells were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde in PBS for one hour, permeabilised with 0.1% Triton X-100/PBS for 10 min and blocked with 2% BSA/PBS for 2 hr. Primary antibodies were diluted in blocking solution (rat anti-HA clone 3F10, 1:1000; polyclonal rabbit anti-c-myc ref C3956, 1:5000; mouse anti-α-tubulin clone DM1A, 1:1000, all from Sigma-Aldrich). Anti-rat Alexa594, anti-mouse Alexa488, anti-rabbit Alexa 488, Anti-rabbit Alexa594 were used as secondary antibodies together with DAPI (all from Life technologies, Switzerland), all diluted 1:1000 in blocking solution. Confocal images were acquired with a LSM700 or a LSM800 scanning confocal microscope (Zeiss).

Fang H., Klages N., Baechler B., Hillner E., Yu L., Pardo M., Choudhary J, & Brochet M. (2017). Multiple short windows of calcium-dependent protein kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis. eLife, 6, e26524.

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Antibody Detection and Chemical Screening

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Europium (Eu) cryptate-or d2-conjugated mouse monoclonal antibodies anti-c-myc (anti-myc-Eu #61MYCKLA or anti-myc-d2 #61MYCDAA), and anti-HA (anti-HA-Eu #610HAKLA or anti-HA-d2 #610HADAA) were purchased from Cisbio (Perkin Elmer). For western and dot blots, the following antibodies were used: rat anti-HA (clone 3F10; Sigma-Aldrich; dilution 1:1000), mouse anti-myc (in-house supernatant from the ECACC hybridoma clone 9E10; 1:50), mouse anti-Flag R ○ (clone M2; Sigma-Aldrich #F1804; 1:1000), rabbit anti-GFP (Cell Signaling #2555; 1:1000), goat HRP-conjugated anti-mouse IgG (Sigma-Aldrich #A4416; 1:5000), donkey HRP-conjugated anti-rat IgG (Jackson Immunoresearch #712-036-153; 1:5000) and goat HRPconjugated TrueBlot R ○ anti-rabbit IgG (Rockland; 1:1000). Protease inhibitors (#P8340), BSA, TX100, Tween 20, DMSO and propidium iodide (#P4864) were from Sigma-Aldrich. KF was from Acros Organics. Hepes was purchased from Thermofisher. The Fr-PPIChem [5] chemical library of 10,314 compounds was provided by the ANR PPIChem consortium (ANR #: ANR-15-CE18-0023). Selected chemical compounds were purchased from Ambinter. All PCR primers were purchased from Sigma-Aldrich or Eurofins ( Suppl. Table 1 ). Restriction enzymes were from New England Biolabs.

Journet A., Barette C., Aubry L., Soleilhac E, & Fauvarque M.O. (2022). Identification of chemicals breaking the USP8 interaction with its endocytic substrate CHMP1B. SLAS discovery : advancing life sciences R & D, 27(7).

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