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Rat anti ha

Manufactured by Merck Group
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Sourced in Germany, France

Rat anti-HA is a monoclonal antibody that specifically binds to the hemagglutinin (HA) tag, a commonly used epitope tag for protein expression and detection. The antibody is derived from rat and can be used in various laboratory applications that require the detection or purification of HA-tagged proteins.

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Lab products found in correlation

Chicken anti gfp, by Abcam (5 mentions) Mouse anti flag, by Merck Group (4 mentions) Mouse anti v5, by Thermo Fisher Scientific (3 mentions) Donkey anti mouse cy3, by Jackson ImmunoResearch (3 mentions) Irdye 800, by LI COR (2 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions) Goat anti rat alexa fluor 647, by Thermo Fisher Scientific (2 mentions) Rabbit anti rfp, by Rockland Immunochemicals (2 mentions) Alexa fluor 488 goat anti chicken, by Thermo Fisher Scientific (2 mentions) Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (2 mentions) Mouse anti gra2, by Abcam (2 mentions) Imagequant las 4000, by GE Healthcare (2 mentions) Rabbit anti c myc, by Thermo Fisher Scientific (2 mentions) Lsm 710 confocal microscope, by Zeiss (2 mentions) Alexa fluor 488 conjugated goat anti rat igg, by Thermo Fisher Scientific (2 mentions) F7425, by Merck Group (2 mentions) Ab 11212363, by Merck Group (2 mentions) Ab 477579, by Merck Group (2 mentions) Odyssey infrared imaging system, by LI COR (2 mentions) Mouse anti flag m2, by Merck Group (2 mentions)

Spelling variants of this product

Anti-HA (475 citations) Rat anti-HA (3F10, (9 citations) Rat anti-HA clone 3F10 (6 citations) Rat anti-HA antibody (4 citations) Rat monoclonal anti-HA (3 citations)

41 protocols using rat anti ha

1

Immunofluorescence Staining of Cultured Cells

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Cells were grown on cover slips in 24 well plates and fixed with 4% (v/v) paraformaldehyde for 10 min at room temperature (RT). After several rinses with PBS, samples were blocked for 1 h at RT in blocking solution (CT: 0.5% (v/v) Triton X-100, 5% (v/v) ChemiBLOCKER (Merck, Darmstadt, Germany)). Subsequently, samples were incubated with primary antibodies (rat anti-HA (Roche/Sigma-Aldrich/Merck) dilution 1:100; mouse anti-CNG [23 (link)] dilution 1:200) in CT at 4 °C over night, rinsed for several times with PBS and then incubated with secondary antibodies (goat anti-rat-Alexa488 (1:500), Invitrogen/Thermo Fisher Scientific, Dreieich, Germany (#A11006); donkey anti-mouse-Cy3 (1:400), Dianova, Hamburg, Germany (715-165-150)) in CT at RT for 1 h. Nuclei were stained with TOPRO-3 (dilution 1:1000; Invitrogen/Thermo Fisher Scientific). Finally, samples were washed with PBS, before mounting the coverslips containing cells in Aqua-Poly/Mount (Polysciences, Eppelheim, Germany) on microscope slides. Fluorescent images were obtained with an inverted confocal microscope (TCS SP5II; Leica, Wetzlar, Germany).
Blenau W., Bremer A.S., Schwietz Y., Friedrich D., Ragionieri L., Predel R., Balfanz S, & Baumann A. (2022). PaOctβ2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana. International Journal of Molecular Sciences, 23(3), 1677.
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2

Immunoblotting of Parasitic Proteins

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Parasites were filtered and collected by centrifugation at 1500 × g for 10 min. Samples were lysed with RIPA lysis buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and 50 mM Tris, pH 8.0) for 15 min on ice. Samples were then resuspended with 4X SDS loading dye with 5% w/v beta-mercaptoethanol, boiled at 95 °C for 5 min and separated on a 10% SDS-PAGE gel for 1.2 h at 160 v. EZ-Run Prestained protein (Fisher) ladder was used as a molecular weight marker. Proteins were then semi-dry transferred to nitrocellulose membrane in Towbin buffer (0.025 M Tris, 0.192 M Glycine, 10% methanol) for 30 min at 190 mA and blocked at room temperature in 5% milk in 0.1% Tween/PBS. Blots were then stained with the following antibodies for 1 h at room temperature: rat anti-HA (Merck) at 1:500 and rabbit anti-Tom4059 (link) at 1:2000 or guinea pig anti-CDPK189 (link) at 1:10,000 followed by secondary fluorescent antibodies: goat anti-rat coupled to IRDye 800 (1:10,000, LI-COR) and goat anti-rabbit coupled to IRDye 680CW (1:10,000, LI-COR) and visualised using the Odyssey LCX. MIT-HA could not be imaged on the LI-COR system but was stained using rat anti-HA followed by anti-rat-HRP (1:5000, Abcam, ab6734)) and detected using Pierce ECL Western Blotting Substrate (Thermo Scientific).
Aghabi D., Sloan M., Gill G., Hartmann E., Antipova O., Dou Z., Guerra A.J., Carruthers V.B, & Harding C.R. (2023). The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii. Nature Communications, 14, 3659.
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3

Chick and Mouse Spinal Cord Analysis

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Spinal cords were fixed in 4% paraformaldehyde and then cry-protected in 20% sucrose. To detect effective knockdown of Daam2 and PIP5K in the chick, we generated mRNA probes and performed in situ hybridization (Lee and Deneen, 2012 (link)). For chick spinal cord immunohistochemistry, the following antibodies were used: mouse anti-Pax7 (DSHB), anti-Nkx2.2 (DSHB), anti-Myc (Sigma), and rat anti-HA (Sigma). Mouse spinal cord was analyzed using in situ hybridization: MBP, PLP, PDGFRα; and immunohistochemistry: chick anti-beta galactosidase (Abcam 1:1,000), mouse anti-MBP (Covance 1:500), mouse anti-PLP (1:500), rabbit anti-Olig2 (Abcam 1:1,000), rabbit anti-GFAP (Dako 1:1,000), rabbit anti-Iba-1 (Wako 1:1,000).
Lee H.K., Chaboub L.S., Zhu W., Zollinger D., Rasband M.N., Fancy S.P, & Deneen B. (2015). Daam2-PIP5K Is a Regulatory Pathway for Wnt Signaling and Therapeutic Target for Remyelination in the CNS. Neuron, 85(6), 1227-1243.
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4

Immunostaining of Engineered Cultured Neurons

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Cultured hippocampal neurons were created from WT P0 pups as described above. Neurons were infected at DIV0–DIV1 with AAV-U6-CARMIL3Ctermsg1-HITI-smFP-P2A-mCherry-SynI-Cre and also either infected with pAAV-pMecp2-SpCas9-spA (AAV-Cas9), PX551, or lipofectamine transfected at DIV2 with PX330 (Cas9). At DIV14–16, neurons were fixed in and prepped for primary antibody incubation as described above. Neurons were incubated in primary antibodies: Rat Anti-HA (Sigma, 3F10, 1:200), Chicken Anti-GFP (Abcam, ab13970, 1:1000), and Rabbit anti-RFP (Rockland, 600-041-379, 1:1000) for 16–18 h at 4 °C. Neurons were then washed three times in PBS for 5min each and incubated in secondary antibodies: Alexa Fluor 647 Goat anti-Rat (ThermoFisher, A-21247, 1:1000), Alexa Fluor 488 Goat anti-Chicken (ThermoFisher, A-11006, 1:1000), and Alexa Fluor 555 Goat anti-Rabbit (ThermoFisher, A32732, 1:1000) for 1–2 h. Coverslips were then mounted with mounting media (Fluorsave Reagent, EMD Millipore, 345789) and imaged on a Zeiss 880 Airyscan inverted confocal microscope.
Spence E.F., Dube S., Uezu A., Locke M., Soderblom E.J, & Soderling S.H. (2019). In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation. Nature Communications, 10, 386.
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5

Visualizing Mitochondrial Morphology and Dopaminergic Neurons in Fly Tissues

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For immunohistochemical analysis of mitochondrial morphology of adult fly brains and muscle tissues, 5-day old male flies from 29°C were assayed. In muscle staining, at least 5 individuals were examined for each genotype and the representative images were presented. For analysis of DA neuron number in the various genetic backgrounds, 21-day old male flies were assayed. In DA neuron staining, at least 7 individuals were examined for each genotype. Dissected tissue samples were briefly washed with 1 × PBS and fixed with 4% formaldehyde in 1 × PBS containing 0.25% Triton X-100 for 30 minutes at room temperature. Fixatives were subsequently blocked with 1 × PBS containing 5% normal goat serum and incubatedfor 1 hour at room temperature followed by incubation with primary antibodiesat 4°Covernight.The primary antibodies used were: chicken anti-GFP (1:5,000, Abcam), rat anti-HA (1:1,000; Sigma), rabbit anti-TH (1:1000, Pel-Freez), mouse anti-C-I30 (1:1,000, Abcam) and mouse anti-ATP5a (1,1000, Abcam). After three washing steps with 1× PBS/0.25% Triton X-100 each for 15 minutes at room temperature, the samples were incubated with Alexa Fluor® 594-conjugated, Alexa Fluor® 488-conjugated, and Alexa Fluor® 633-conjugated secondary antibodies (1:500, Molecular Probes) for 3 hours at room temperature and subsequently mounted in SlowFade Gold (Invitrogen).
Wu Z., Tantray I., Lim J., Chen S., Li Y., Davis Z., Sitron C., Dong J., Gispert S., Auburger G., Brandman O., Bi X., Snyder M, & Lu B. (2019). MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure. Molecular cell, 75(4), 835-848.e8.
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6

Western Blot Analysis of Toxoplasma Antigens

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Proteins were separated by SDS/PAGE and transferred to a PVDF membrane. Membranes were blocked for 1 h in TBST + 5% milk, followed by overnight incubation at 4 °C with primary antibody in blocking solution. The next day, membranes were washed 3× with TBST, followed by incubation at room temperature for 1–2 h with HRP-conjugated secondary antibody (Sigma) in blocking buffer. After three washes with TBST, Western blots were imaged using ECL Plus reagent (Pierce) on a GE ImageQuant LAS4000. Antibodies used in this study include: mouse anti-GRA1 (1:1,000 dilution; BioVision), mouse anti-GRA2 (1:1,000 dilution; BioVision), mouse anti-GRA3 (gift of J.-F. Dubremetz, University of Montpellier, Montpellier, France; 1:2,000 dilution), mouse anti-GRA4 (gift of L. D. Sibley; 1:2,000 dilution), mouse anti-GRA5 (1:1,000 dilution; BioVision), mouse anti-GRA6 (gift of L. D. Sibley; 1:4,000 dilution), rabbit anti-GRA7 (gift of L. D. Sibley; 1:5,000 dilution), rabbit anti-ROP2 (1:10,000 dilution), rat anti-HA (1:1,00 dilution; Sigma). Western blots for quantification were performed independently with each antibody to avoid residual signal due to incomplete stripping.
Beraki T., Hu X., Broncel M., Young J.C., O’Shaughnessy W.J., Borek D., Treeck M, & Reese M.L. (2019). Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole. Proceedings of the National Academy of Sciences of the United States of America, 116(13), 6361-6370.
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7

Purification and Western Blot Analysis of Plasmodium Proteins

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P. falciparum parasites were released from erythrocytes by treatment with 0.15% saponin in PBS. Murine erythrocytes infected with the pv5-tag-GFPPV or exp2-mCherry P. berghei lines (62 (link)) were purified on a Nycodenz gradient and lysed hypotonically for 1 h on ice in 10 mM Tris⋅HCl, pH 7.5. P. berghei lysates were centrifuged for 50 min at 100,000 × g. Membrane pellets were resuspended in 0.1 M Na2CO3 in PBS or in 1% Triton X-100 in PBS, respectively, and centrifuged for 50 min at 100,000 × g. Proteins were separated on SDS-polyacrylamide and transferred to nitrocellulose membranes. Western blotting was performed using rat anti-mCherry (1:5,000; ChromoTek), chicken anti-GFP (1:5,000; Abcam), rat anti-HA (1:1,000; Sigma Aldrich), rat anti-PfBiP (1:1,000) (63 (link)), and rabbit anti-human hemoglobin α-primary antibodies (1:1,000; Abcam) followed by chemiluminescence detection with horseradish peroxidase-coupled secondary antibodies (1:10,000; Sigma Aldrich, or 1:5,000; Jackson ImmunoResearch).
Matz J.M., Drepper B., Blum T.B., van Genderen E., Burrell A., Martin P., Stach T., Collinson L.M., Abrahams J.P., Matuschewski K, & Blackman M.J. (2020). A lipocalin mediates unidirectional heme biomineralization in malaria parasites. Proceedings of the National Academy of Sciences of the United States of America, 117(28), 16546-16556.
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8

Immunoblot Analysis of Parasite Proteins

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Total parasite lysates (collected at 1500 g, 10 min, RT and lysed in 1x sample buffer), or immunoprecipitation fractions (105 or 107 parasites per lane respectively) were separated by SDS-PAGE and used for immunoblot analyses. After blocking in Odyssey block (LI-COR Biosciences) or in 1X TBS, Tween-0.2%, 5% BSA, blots were probed with: monoclonal mouse anti-ATrx1 11G8 at 1:5000 [9 (link)]; mouse anti-HA mAb at 0.1 μg/mL (Covance); rat anti-HA (Sigma-Aldrich, 1:50); Mouse-anti-His (Amersham (GE), 1:1000); rabbit anti-c-Myc (Thermo Scientific, 1:1,000); rabbit anti-Mic5 (gift of Dr. Vern Carruthers, 1:10,000) and anti-actin. This was followed by goat anti-mouse Ig coupled to IRDye 800 (1:10,000, LI-COR) or goat anti-rabbit Ig coupled to IRDye 680 (1:10,000, LI-COR), or goat anti-rabbit or anti-mouse HRP conjugated (Promega, 1:10,000).
Biddau M., Bouchut A., Major J., Saveria T., Tottey J., Oka O., van-Lith M., Jennings K.E., Ovciarikova J., DeRocher A., Striepen B., Waller R.F., Parsons M, & Sheiner L. (2018). Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii. PLoS Pathogens, 14(2), e1006836.
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9

Staining and Imaging Notch Signaling

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Wing discs from wandering larvae or ovaries and brain from adult flies were dissected in 1x PBS and fixed for 30 minutes in 4% paraformaldehyde in PBS. Tissues were then washed with 0.2% PBST for total Notch staining or with cell-culture grade PBS (Thermo Fisher Scientific, #10010023) for extracellular Notch staining. All antibodies used in this study are listed in S1 Table. Primary antibodies [mouse anti-Notch intracellular domain (NICD) (1:50; DSHB, C17.9C6), mouse anti-Cut (1:100; DSHB, 2B10), rat anti-HA (1:100, Sigma-Aldrich, 11867423001), or mouse anti-NECD (1:100; DSHB, C458.2H)] were applied in 0.2% PBST or PBS with 5% NDS/0.1% NaN3 overnight at 4°C. Tissue was then washed with 0.2% PBST or PBS (3 times, 15 minutes each) and secondary antibodies/stains [donkey anti-rat IgG-Cy3 (1:500; Jackson ImmunoResearch #712-165-153), donkey anti-mouse IgG-Alexa-647 (1:500; Jackson ImmunoResearch #715-605-151), donkey anti-mouse Alexa-488 (1:500; Jackson ImmunoResearch #715-545-151), and/or Alexa-488 Phalloidin (1:1000; ThermoFisher A12379)] were applied in 0.2% PBST+NDS or PBS+NDS for 2 hours at room temperature. Tissues were further washed with 0.2% PBST or PBS and mounted in Vectashield with DAPI (Vector labs). Images were taken with LSM 710 Confocal Microscope (Zeiss).
Salazar J.L., Yang S.A., Lin Y.Q., Li-Kroeger D., Marcogliese P.C., Deal S.L., Neely G.G, & Yamamoto S. (2021). TM2D genes regulate Notch signaling and neuronal function in Drosophila. PLoS Genetics, 17(12), e1009962.
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10

Immunofluorescence Assay for Parasite Analysis

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Parasites were grown within HFF on coverslips. Immunofluorescence assays were carried out as indicated previously ([14 (link)] or [58 (link)]). Antibodies and concentrations used were: rat anti-HA (Sigma-Aldrich, 1:50); rabbit anti-c-Myc (Thermo Scientific, 1:1,000); rabbit anti-CSP60 (Reff, 1:1,000); FITC-coupled rat anti-HA (Roche, 3 μg/mL); rabbit anti-IMC1 (gift of Con Beckers, 1:1,000); in Fig 3 the marker for the apicoplast stroma was the naturally biotinylated apicoplast luminal protein acetyl CoA carboxylase revealed by Texas Red coupled-streptavidin (Invitrogen, 1 μg/mL) [59 (link)]. Secondary antibodies: Cy2 goat anti-rabbit, Cy3 goat anti-Rabbit, Cy2 goat anti-mouse, Cy3 goat anti-mouse (all Jackson Immuno Research Laboratories, 1:2,000).
Biddau M., Bouchut A., Major J., Saveria T., Tottey J., Oka O., van-Lith M., Jennings K.E., Ovciarikova J., DeRocher A., Striepen B., Waller R.F., Parsons M, & Sheiner L. (2018). Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii. PLoS Pathogens, 14(2), e1006836.
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