Infected cells grown on glass coverslips were fixed using methanol at –20°C for 20 min or 4% formaldehyde at room temperature (RT) for 20 min, as stated in the text. Methanol-fixed samples were washed three times for 5 min with PBS and blocked using 3% bovine serum albumin (BSA) in PBS for 1 h at RT. Formaldehyde-fixed samples were rinsed once with PBS, permeabilized with 0.2% Triton X-100 (TTX-100) for 20 min and then blocked as described above. GRA16HA (and other hemagglutinin [HA]-tagged proteins) was detected using rat anti-HA antibodies (Roche), while GRA24MYC was detected using rabbit anti-MYC tag antibody 9E10 (Santa Cruz Biotechnology). This anti-MYC tag antibody does not detect host c-Myc. Host c-Myc was detected using monoclonal antibody Y69, which does not cross-react with the MYC tag expressed by GRA24MYC. Primary antibodies were detected with goat polyclonal Alexa Fluor-conjugated secondary antibodies (Invitrogen). Vectashield with 4′,6′-diamidino-2-phenylindole (DAPI) stain (Vector Laboratories) was used to mount the coverslips on slides. Fluorescence was detected using a LSM710 inverted confocal microscope (Zeiss) or epifluorescence microscope as stated in the text. Images were analyzed using ImageJ. All images shown for any given condition/staining in any given comparison/data set were obtained using identical parameters.
Rat anti ha antibody
Manufactured by Roche
Sourced in United States, Germany
The Rat anti-HA antibody is a laboratory reagent used to detect and identify proteins tagged with the HA (Hemagglutinin) epitope. It is a monoclonal antibody derived from rats, which specifically binds to the HA tag, allowing for the identification and isolation of HA-tagged proteins in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Lsm 710 inverted confocal microscope, by Zeiss (2 mentions)
Rabbit anti gfp antibody, by Abcam (2 mentions)
Clarity or clarity max substrate, by Bio-Rad (2 mentions)
Protein g coated magnetic beads, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin antibody, by Merck Group (2 mentions)
Mouse monoclonal antibody to β actin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mouse anti myc antibody, by Santa Cruz Biotechnology (1 mentions)
Alkaline phosphatase kit, by Promega (1 mentions)
Vectashield with dapi stain, by Vector Laboratories (1 mentions)
710 confocal microscope, by Zeiss (1 mentions)
Fibrillarin, by Abcam (1 mentions)
Mouse anti gfp antibody, by Roche (1 mentions)
Mouse anti calnexin, by BD (1 mentions)
A1 laser scanning microscope, by Nikon (1 mentions)
Mouse anti gm130, by BD (1 mentions)
Ecl detecting reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc machine, by Bio-Rad (1 mentions)
Hrp conjugated donkey anti rabbit antibody, by GE Healthcare (1 mentions)
41 protocols using rat anti ha antibody
1
Immunofluorescence Staining of Toxoplasma Proteins
2
Western Blot Detection of Parasite Proteins
3
Immunofluorescence Staining of Infected Cells
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Infected cells grown on glass coverslips were fixed using methanol at -20°C for 20 min or 3–4% formaldehyde at room temperature (RT) for 15 min, as stated in the text. Methanol-fixed samples were washed three times for 5 min with PBS and blocked using 3% BSA in PBS for 1h at RT. Formaldehyde-fixed samples were rinsed once with PBS, permeabilized with 0.2% Triton-X 100 (TTX-100) for 20 min, and then blocked as described above. MYR1(N) protein was detected with mouse anti-MYR1 antibodies [27 (link)], while MYR1(C) protein was detected with rat anti-HA antibodies or mouse anti-MYR1 primary antibodies. GRA7 protein was detected using rabbit anti-GRA7 antibodies [34 (link)]. GRA16-HA (and other HA-tagged proteins) was detected using rat anti-HA antibodies (Roche) while GRA24-Myc was detected using rabbit anti-Myc tag antibodies (Cell Signaling Technologies). Primary antibodies were detected with goat polyclonal Alexa Fluor-conjugated secondary antibodies (Invitrogen). Vectashield with DAPI stain (Vector Laboratories) was used to mount the coverslips on slides. Fluorescence was detected using a LSM710 inverted confocal microscope (Zeiss) or epifluorescence microscope, as stated in the text. Images were analyzed using ImageJ. All images shown for any given condition/staining in any given comparison/dataset were obtained using identical parameters.
4
Immunoprecipitation and Western Blot Analysis
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HEK-293T cells were harvested 24h post-transfection. For the production of whole-cell lysates, cells were lysed in 300 μl of lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl , 1mM GTP, 10mM MgCl2, a protease inhibitor cocktail [1x EDTA-free protease inhibitors, Sigma], 10mM NaF, 1mM Na3VO4, 2μM Bortezomib) at 4°C. 60 μl of lysates was saved as input and the rest was subject to immunoprecipitation.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
5
Immunoprecipitation and Western Blot Analysis
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HEK-293T cells were harvested 24h post-transfection. For the production of whole-cell lysates, cells were lysed in 300 μl of lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl , 1mM GTP, 10mM MgCl2, a protease inhibitor cocktail [1x EDTA-free protease inhibitors, Sigma], 10mM NaF, 1mM Na3VO4, 2μM Bortezomib) at 4°C. 60 μl of lysates was saved as input and the rest was subject to immunoprecipitation.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
6
Immunofluorescence and FISH Protocols for Drosophila Embryos and Ovaries
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Immunofluorescence and FISH were performed on embryos and ovaries as described in references [4] (link), [83] (link). Polytene chromosomes were dissected in 0.7% NaCl, squashed, and fixed in 1.8% PFA, 45% acetic acid for 17 minutes. They were then washed in 1% Triton X in PBS for 10 minutes, then washed in 5% milk in PBS for 1 hour, incubated with primary antibody overnight at 4°C, washed in 5% milk in PBS for 10 minutes, incubated with secondary antibody for 1 hour at room temperature, and then washed for 10 minutes in buffer A (0.15M NaCl, 0.2% NP40 substitute, 0.2%Tween 20) followed by 10 minutes in buffer B (0.20M NaCl, 0.2% NP40 substitute, 0.2%Tween 20).
Rat anti-HA antibody (Roche, 3F10) was used at 1∶100, rat anti-Vasa (DSHB) was used at 1∶25, Fibrillarin (Abcam, Ab5281) was used at 1∶100, anti-HP1a antibody (C1A9, DSHB) was used at 1∶100. Alexa fluorophore-conjugated secondary antibodies were used to detect the primary antibody. Fluorescently labeled probes against GA-rich satellites, AACAC, 2L3L, 359 bp and dodeca were obtained from Sigma with sequences described in references [8] (link), [83] (link), [97] . Imaging was carried out using a Zeiss 710 confocal microscope at Cornell University's Microscopy and Imaging Facility.
Rat anti-HA antibody (Roche, 3F10) was used at 1∶100, rat anti-Vasa (DSHB) was used at 1∶25, Fibrillarin (Abcam, Ab5281) was used at 1∶100, anti-HP1a antibody (C1A9, DSHB) was used at 1∶100. Alexa fluorophore-conjugated secondary antibodies were used to detect the primary antibody. Fluorescently labeled probes against GA-rich satellites, AACAC, 2L3L, 359 bp and dodeca were obtained from Sigma with sequences described in references [8] (link), [83] (link), [97] . Imaging was carried out using a Zeiss 710 confocal microscope at Cornell University's Microscopy and Imaging Facility.
7
Antibody Staining of Malaria Proteins
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The following primary antibodies and antisera were used: rat anti-HA antibody (Roche, Basel, Switzerland); mouse anti-GFP antibody (Roche); mouse polyclonal antisera against Pf39 [56 (link)]; rabbit polyclonal antisera against Pfs230 [57 (link)]; mouse polyclonal antisera against PfFalcilysin [58 (link)]; rabbit anti-H3K4me3 antibody (abcam, Cambridge, UK), rabbit anti-H3K18me1 antibody (AB clonal, Cummings Park, W). The generation of polyclonal mouse antisera against Pf92 is described below.
8
Immunofluorescence Localization of ASIC2 Isoforms
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For immunofluorescence, CHO cells were initially transfected with HA-tagged ASIC2a or ASIC2b together with a membrane-targeted Lck-GFP in 35 mm dishes and re-plated into 4 well chamber glass slides 1 day after transfection. Similar to what has been describe earlier [54 (link)], ASIC2 was detected with a rat anti-HA antibody (Roche, #11867423001, 1:1000 dilution). ER and Golgi was detected with a mouse anti-calnexin (BD biosciences, #610523, 1:500 dilution) and a mouse anti-GM130 (BD biosciences, #610822, 1:500 dilution), respectively. Secondary antibodies used were Dylight 568-conjugated donkey anti-rat and Dylight 649-conjugated donkey anti-mouse antibodies. Confocal images were captured using a Nikon A1 laser scanning microscope. Illumination was provided by an argon (Ar, 458, 488, 514 nm lines) and two diode (561 and 640 nm lines) lasers. Green, red and far red channels were imaged sequentially. Images were captured with a 63×/1.2NA PL APO water lens. Images were exported and further processed with Adobe Photoshop as described previously [51 (link)].
9
Immunoblotting for Virus-Expressed GFP and pCas13a
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Immunoblotting was performed as reported previously [26 (link)]. Briefly, 100 mg of ground tissue was used for protein extraction. Total proteins were resolved on 10% SDS page. For blotting, rabbit anti-GFP antibody (abcam, Cambridge, UK) (for virus-expressed GFP detection) or rat anti-HA antibody (Roche, Darmstadt, Germany) (for pCas13a detection) at 1:3000 or in 1:1000 dilutions, respectively, were used. All blots were treated with their respective secondary antibodies. ECL detecting reagent from Thermo Scientific was used to detect the signal using a ChemiDoc machine (Bio-Rad, Hercules, CA, USA).
10
Detection of AP2IX-4 in Parasite Lysates
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Immunoblotting of parasite lysate was used to detect AP2IX-4 in various clones by virtue of the 3xHA epitope tag. Parasite lysate was prepared in lysis buffer (150 mM NaCl, 50 mM Tris-Cl [pH 7.5], 0.1% NP-40) and sonicated prior to resolution on SDS-PAGE and transfer to a nitrocellulose membrane. HA epitopes were detected using rat anti-HA antibody (Roche) at a 1:5,000 dilution followed by secondary probing with horseradish peroxidase (HRP)-conjugated goat anti-rat antibody (GE Healthcare) at a 1:2,000 dilution. To ensure equal loading of samples, we also probed with rabbit anti-β-tubulin antibody (kindly supplied by David Sibley, Washington University) at a 1:2,000 dilution followed by HRP-conjugated donkey anti-rabbit antibody (GE Healthcare) at a 1:2,000 dilution.
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