All custom made antibodies were prepared in rabbits by Eurogentec (Eurogentec SA). Rabbit antisera were produced against DRM2 peptides EP112214 (NSDDEKDPNSNENGS) and EP112215 (ESKGEPRSSVDDEPI) following their double‐X immunization program and then affinity‐purified on EP112215. Antibodies for NRPD1 detection were also raised in rabbits against EP112201 (ESKGEPRSSVDDEPI) peptide and affinity purified by Eurogentec. His‐tagged UAP56 protein was produced from pET‐28a‐UAP56 in BL21 E. coli strain and purified with His‐bind resin following the supplier's instructions (Millipore). Anti‐UAP56 serum was then produced in rabbits using this recombinant protein as antigen. Anti‐AGO4 antibodies were previously used by Lahmy et al. 19 . Monoclonal antibody 8WG16 (ab817; Abcam) was used to detect NRPB1; histone H3 (ab1791; Abcam) and UGPase polyclonal antibodies (AS05 086, Agrisera) were also used for nucleus and cytoplasm controls. Affinity‐purified anti‐HA antibodies coupled to HRP (Sigma‐Aldrich, clone HA‐7) were used to detect DRM2 in transgenic tagged lines.
Clone ha 7
Manufactured by Merck Group
The Clone HA-7 is a laboratory equipment product designed for the isolation and purification of specific proteins or molecules from complex biological samples. It functions as an affinity chromatography tool, utilizing a monoclonal antibody specific to the target of interest to capture and separate the desired component from the sample mixture.
Automatically generated - may contain errors
Lab products found in correlation
His bind resin, by Merck Group (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab817, by Abcam (1 mentions)
As05086, by Agrisera (1 mentions)
Yeast strain ah109, by Takara Bio (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Pbridge, by Takara Bio (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Clone 9e10, by Merck Group (1 mentions)
Clone m2, by Merck Group (1 mentions)
Ca 630, by Roche (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Mouse immunoglobulin g igg, by Merck Group (1 mentions)
Prestained protein molecular weight marker, by Thermo Fisher Scientific (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha clone ha 7, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 633, by Thermo Fisher Scientific (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
8 protocols using clone ha 7
1
Antibody Generation and Characterization
2
Yeast Trehalose-6-Phosphate Phosphatase Mutants
3
Yeast-based Interactome Mapping of Maize Transcription Regulators
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Yeast codon-optimized ORFs of CT2 (GRMZM2G064732), CT2CA, ZmXLG1 (GRMZM2G127739), ZmXLG3a (GRMZM2G016858), and ZmXLG3b (GRMZM2G429113) were cloned between the EcoRI and XhoI restriction sites of MCS1 of pGADT7 (Clontech). ZmGB1 (GRMZM2G045314) was cloned between the EcoRI and BamHI restriction sites of MCS1. ZmRGG2 (GRMZM6G935329) was cloned between the NotI and BglII restriction sites of MCS2 of pBRIDGE (Clontech), respectively. The primer sequences are shown in the supplementary information. The yeast assay was performed in the AH109 yeast strain (Clontech). The double transformants were selected on SC -Trp -Leu (-LW) plates. The interaction was tested on the SC -Trp -Leu -His (-LWH) medium supplemented with 1 mM 3-Amino-1,2,4-triazole (3-AT) to suppress histidine synthesis. The HA-tag was detected using the monoclonal anti-HA antibody produced in mouse (Sigma, clone HA-7).
4
Co-immunoprecipitation of Protein Complexes
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For co-immunoprecipitation (co-IP), cell lysates were prepared by adding lysis buffer (150 mM NaCl, 1% IGEPAL® CA-630, 50 mM Tris·Cl; pH 8.0) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The lysate was immunoprecipitated using 2–3 μg of antibody (specificity indicated in the figures), mouse immunoglobulin G (IgG; Sigma-Aldrich, St. Louis, MO, USA), and incubated with 50 μL of Protein G-Sepharose (GE Healthcare, Chicago, IL, USA). The immunoprecipitates were washed three times in 1 mL of ice-cold lysis buffer, followed by additional wash an additional time with 1 mL of 50 mM Tris·Cl (pH 8.0). The precipitated proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8%–12%). For western blot analysis, the blots were incubated using the antibody indicated in the figures. All co-IPs and western blot analyses were performed more than twice to confirm that the data were reproducible. The following antibodies were used in the co-IPs and western blot analyses: monoclonal anti-FLAG antibody (1:2000, Clone M2; Sigma-Aldrich), monoclonal anti-HA antibody (1:2000, Clone HA-7; Sigma-Aldrich), and monoclonal anti-Myc antibody (1:2000, Clone 9E10; Sigma-Aldrich).
5
Spermatium Counting and Western Blot Analysis
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Spermatium counting was performed as follows: each strain was grown on M2 medium at 27°C for 21 days. To collect spermatia, cultures were washed with 1.5 mL of 0.05% Tween 20 in sterile water. Numeration proceeded through Malassez counting chamber. Grafting was assayed as in [41 (link)]. Western blot analyses were performed on perithecia grown for two days, as in [109 (link)]. We used the anti-HA high affinity monoclonal antibody from mouse to recognize the HA-peptide (clone HA-7, ref H3663, Sigma-Aldrich). Detection was performed using Chromogenic Western Blot Kit (Anti-Mouse, EnzoLife, ENZ-KIT182), molecular scale was given by the Prestained Protein Molecular Weight Marker (Thermo Scientific, 11583100)
6
Immunoprecipitation and Western Blot Analysis of VgrG Proteins
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HEK293 Flp-In T-REx cells carrying the genes for vgrG2aHA-Strep or vgrG2bHA-Strep were cultured in 10-cm petri dishes in Dulbecco’s modified Eagle’s medium (DMEM) according to the manufacturer’s instructions (Invitrogen). The expression of recombinant vgrG2a or vgrG2b was induced by adding doxycycline to the medium to 1 µg/ml for 16 h. Subsequently, cells were scraped off the culture dish and centrifuged, and after two rinses with PBS, cells were lysed with 75 µl/dish of 50 mM HEPES, 100 mM KCl, 1 mM EDTA, 1 mM MgCl2, 1% NP-40, 1 mM β-mercaptoethanol, and a cocktail of protease inhibitors (Roche). Six microliters of ascites fluid containing monoclonal antibody against the HA tag was added (clone HA-7; Sigma-Aldrich), and following incubation for 30 min on ice, immunoprecipitation was performed by adding 50 µl of protein G coupled to magnetic beads (Dynabeads, Invitrogen). Following several rinses, bound protein was eluted from the beads with 40 µl of Laemmli gel electrophoresis buffer containing 2% SDS. Eluted proteins, as well as cell lysates before and after immunoprecipitation, were analyzed by Western blotting. Western blots were probed with mouse-anti-HA (clone HA-7; Sigma-Aldrich), and rabbit antibodies against γ-tubulin or GCP4, respectively (53 (link)).
7
Lassa Virus Immunodetection Assays
8
Kinase Activity Assay for Itk Mutants
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