Unless indicated, all chemicals and drugs were from Sigma-Aldrich/Merck Millipore. Primary antibodies used in this study included the following: Rabbit anti-HA clone C29F4, anti-pSrc Tyr416 clones 100F9 and D49G4, anti-Na+/K+ ATPase #3010, and anti-GFP clone D5.1 (Cell Signaling Technology); rabbit Anti-APP clone Y188 (Abcam); mouse anti-HA clone 16B12 (Covance); rabbit anti-actin #AAN01 (Cytoskeleton Inc); anti-transferrin receptor clone H68.4 (Thermo Fisher Scientific); rabbit anti-methylated Lysine (Enzo Life Sciences); mouse anti-Fyn clone 25 #610163, anti-flotillin-1 clone 18, and anti-PP2Acα clone 46 (BD Transduction Laboratories); rabbit anti-Fyn clone EPR5500, mouse anti-Bα clone 2G9, anti-actin clone C4, anti-LCMT1 clone 4A4, anti-APP clone 22C11 (MAB348), and anti-demethyl PP2Ac clone 1D6 (Merck Millipore).
Rabbit anti ha clone c29f4
Manufactured by Cell Signaling Technology
Rabbit anti-HA clone C29F4 is a primary antibody that specifically recognizes the hemagglutinin (HA) epitope tag. This antibody is produced in rabbits and can be used for the detection and immunopurification of HA-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti fyn clone epr5500, by Merck Group (2 mentions)
Mouse anti bα clone 2g9, by Merck Group (2 mentions)
Mouse anti ha clone 16b12, by Fortrea (2 mentions)
Mouse anti cmyc clone 9e10, by Merck Group (2 mentions)
Iblot system, by Thermo Fisher Scientific (2 mentions)
Anti transferrin receptor clone h68, by Thermo Fisher Scientific (1 mentions)
Anti actin clone c4, by Merck Group (1 mentions)
Donkey anti mouse hrp, by Thermo Fisher Scientific (1 mentions)
Mes buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha clone 12ca5, by Roche (1 mentions)
Anti human pd 1 clone eh12.2h7, by BioLegend (1 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (1 mentions)
Deltavision microscopy system, by Cytiva (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Mouse anti p44 42 mapk erk1 2 l34f12, by Cell Signaling Technology (1 mentions)
Mouse anti gsk3β clone 3d10, by Cell Signaling Technology (1 mentions)
7 protocols using rabbit anti ha clone c29f4
1
Antibody Characterization for Protein Studies
2
Immunoblot Analysis of Parasite Proteins
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For immunoblot analysis, parasites were grown in the absence and presence of ATc (1 mg/ml) for 48–50 h. Approximately 3 × 106 freshly released parasites were harvested and resuspended in reducing sample buffer. Equal amounts of protein lysate were separated using NuPAGE 4–12% Bis-Tris gels in MES buffer (Life Technologies) and transferred onto nitrocellulose membranes using the iBlot system (Life Technologies). Membranes were blocked in PBS with 5% skim milk (wt/vol) for 1 h before incubation with one of the primary antibodies (in blocking solution) rabbit anti-HA clone C29F4 (Cell Signaling Technology), 1:4000; mouse anti-cMyc clone 9E10 (Sigma-Aldrich), 1:1000; rabbit anti-Act239-253 (Angrisano et al., 2012 (link)), 1:4000; and rabbit anti-TgADF (kindly provided by D. Sibley, Washington University, St. Louis), 1:4000. Secondary antibodies used were goat anti-rabbit horseradish peroxidase (HRP) and donkey anti-mouse HRP (Life Technologies) in 1:4000 and 1:2000 dilutions, respectively. Gels were run in duplicate, and membranes were stripped, blocked, and reprobed for multiple analyses.
3
Invasion Assay for Toxoplasma gondii
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MIC7 iKO parasites were treated with DMSO or rapamycin and were prepared as described for the video recording assays. Approximately 8E5 tachyzoites in 300 μL of motility buffer were deposited on 12 mm glass coverslip previously coated with poly-L-lysine (as above) and placed in a 24 well plate. Tachyzoites were gently centrifuged (300 x g, 3 min) to ensure rapid contact with the host cell (non-fluorescent HFF and GFP-GPI expressing U2OS cells) and allowed to invade for 2 to 4 min periods at 37°C and 5% CO2. Samples were immediately fixed in 3.2% PFA in PHEM pH 7.5 (20 min) prior to be processed for IFA. Blocking step and anti-TgSAG1 staining were performed as for the gliding assay except that the incubation with SAG1 primary antibody and Alexa Fluor 633-conjugated secondary antibody was reduced to 30 min. After SAG1 staining, the HFF-tachyzoite samples were permeabilised with (0.2% Triton X-100/PBS, 5 min) prior to a second step of blocking. MIC7 labelling was performed using rabbit anti-HA (clone C29F4), (1:800, 2 hr; Cell Signaling) followed by Alexa Fluor 488-conjugated HCA anti-rabbit antibody (2 mg/mL stock, 1:800, 2 hr).
4
Immunoblot Analysis of Malaria Parasites
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For immunoblots, infected RBCs were lysed and washed as above. Parasite material was resuspended in 1x SDS sample buffer. Extracted proteins were separated on NuPAGE™ Novex® 4–12 % Bis-Tris protein gels in MES buffer (Life Technologies) and transferred onto nitrocellulose membranes using the iBlot® system (Life Technologies). Rabbit anti-HA (clone C29F4, Cell Signaling) was diluted 1:4,000; mouse anti-HA (clone 12CA5, Roche), mouse anti-GFP (clones 7.1 and 13.1, Roche), rabbit anti-ADF1 [41 (link)], rabbit anti-F-Actin [42 ] and mouse anti-Pfs16 (a kind gift from Professor Robert Sauerwein) were diluted 1:1,000 in 5 % skim milk/PBS (w/vol). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were used as secondary antibodies (Jackson IR) and diluted 1:10,000 in 5 % skim milk/PBS.
5
Monoclonal Antibody Characterization Protocol
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The mouse anti-HA monoclonal antibody (mAb) and the mouse anti-human IgG (29.5; Fc specific; anti-Fc) were generated in our lab and have been previously described (17 (link)). The anti-human IgG (Fc specific) labelled with peroxidase (POD) was from Sigma-Aldrich, the anti-human PD-1 (clone EH12.2H7) from BioLegend, and the anti-mouse IgG-PE and IgG-APC from Jackson ImmunoResearch. The rabbit anti-HA (clone C29F4) and the anti-β-actin (clone C4) mAbs, and the anti-rabbit IgG-POD, and the anti-mouse IgG-HRP, were from Cell Signaling, MP Biomedicals, Promega, and Sigma-Aldrich, respectively. The streptavidin-AF555 and the anti-rabbit IgG (H+L)-biotinylated were from Life Technologies and Jackson Immunoresearch, respectively. The anti-human CD14-APC (clone M5E2) mAb employed to stain surface expression of anti-CD3 single chain fragments, and the anti-mouse CD45-APC (clone 104) mAb used to exclude the TCS in the reporter assays, were obtained from BioLegend.
6
Immunofluorescence Analysis of T. gondii Actin
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For immunofluorescence analysis, HFF cells were grown on coverslips and inoculated with T. gondii parasites in the absence and presence of ATc (at 1 mg/ml) for 48–50 h. To investigate the distribution of actin in extracellular parasites, growth in ATc was extended to 68–72 h, and freshly released tachyzoites were harvested. Cells were fixed with 4% (wt/vol) paraformaldehyde and 0.0075% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in phosphate-buffered saline (PBS) for 10 min before permeabilization with 1% (vol/vol) Triton X-100 in PBS for 10 min, followed by blocking in 3% (wt/vol) bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h. Primary antibodies were diluted in blocking solution and used in the following concentrations: rabbit anti-HA clone C29F4 (Cell Signaling Technology, Danvers, MA), 1:4000; mouse anti-cMyc clone 9E10 (Sigma-Aldrich), 1:2000; and rabbit anti-Act239-253 (Angrisano et al., 2012 (link)), 1:4000. Secondary antibodies used were Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit (Life Technologies) in 1:4000 dilutions.
Fluorescence microscopy was performed using a DeltaVision Microscopy System (Applied Precision, Seattle, WA) and an Olympus UPlanSApo 60×/numerical aperture (NA) 1.4 or 100×/1.4 NA oil objective.
Fluorescence microscopy was performed using a DeltaVision Microscopy System (Applied Precision, Seattle, WA) and an Olympus UPlanSApo 60×/numerical aperture (NA) 1.4 or 100×/1.4 NA oil objective.
7
Antibody-Based Analysis of Protein Signaling
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Unless indicated, all chemicals were purchased from Sigma/Merck Millipore. Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Acα/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) (59 (link)) (a gift from Peter Davies). Mouse (clone C4, Merck Millipore) or rabbit (Cytoskeleton) anti-actin, and mouse (clone 236-10501, Life Technologies Australia Pty Ltd) or rabbit (Epitomics) anti-α-tubulin antibodies were used for protein normalization.
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