FM 4-64 Dye (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide) and IPTG (isopropylthio-β-galactoside) were purchased from Invitrogen. EDTA-free protease inhibitor cocktail, Pefabloc SC (AEBSF), endoglycosidase H, and anti–c-myc antibody were purchased from Roche. DTT (dithiothreitol) was purchased from USB. The anti-Pgk1p and anti-CPY antibodies were purchased from Molecular Probes. The anti-mouse immunoglobulin G (IgG) horseradish peroxidase–conjugated antibody, concanavalin A (C7275), lysozyme, and calcofluor white were purchased from Sigma. The anti-rabbit IgG horseradish peroxidase–linked antibody, Amersham ECL Western Blotting Detection Reagent, and glutathione Sepharose 4B were purchased from GE Healthcare. The anti-Vps74p antibody was from the Banfield lab collection. The anti-coatomer antibody was kindly provided by Anne Spang (University of Basel). The anti-Gas1p antibody was kindly provided by Howard Riezman (University of Geneva, Switzerland). The mNeonGreen coding sequence was kindly provided by Chris Fromme (Cornell University).
Anti c myc antibody
Manufactured by Roche
The Anti–c-myc antibody is a laboratory tool used for the detection and analysis of proteins that contain the c-myc epitope. It is a highly specific and sensitive antibody that can be used in various immunological techniques, such as Western blotting, immunoprecipitation, and immunocytochemistry, to identify and quantify c-myc-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (1 mentions)
Endoglycosidase h, by Roche (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Pgex 4t 1 vector, by GE Healthcare (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Anti ha antibody, by Roche (1 mentions)
3 protocols using anti c myc antibody
1
Protein Purification and Antibody Detection
2
Immunoprecipitation of DOZI and CITH complexes
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Parasites were grown to a parasitemia of below 3% and collected by heart puncture. Gametocytes were enriched by Nycodenz gradient purification as described [10 (link)] and protein lysates prepared by NP-40 (0.5%) rupture on ice in 50 mM NaCl, 150 mM TrisHcl. Immunoprecipitation of DOZI::GFP, CITH::GFP, and GFP were performed with mouse monoclonal anti-GFP antibodies from Roche (catalogue # 11814460001); the control antibody was a mouse monoclonal anti-cmyc antibody (catalogue # 11667149001). Antibody-mRNP complexes were captured on protein G sepharose beads (GE Healthcare) and eluted with 2 × SDS-PAGE loading dye for protein and western analyses, or 1 mL TRIzol for RNA isolation. From the initial lysates one-quarter (50 μL) was kept as input sample, one-quarter was further processed for anti-GFP, one-quarter for anti-c-myc, and one-quarter for beads-only IPs. The nature of the RIP experiment results in minimal RNA co-elution in the control samples, which is why no loading control across the four samples is provided. The differences in output of known translationally repressed mRNAs (that is, p25, p28, and ap2-0) and known translated mRNAs (that is, dozi, cith, and alba-3) act as internal controls of the procedure during RT-PCR.
3
Expression and Purification of SQU and KEW
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Full-length coding sequences of SQU and KEW, and mutant SQU(m G1+G3) and KEW(m G3+G4) with all ATP/GTP binding sites mutated as indicated in Supplemental Table 3, were cloned into the pGEX-4T-1 vector (GE Healthcare), and transformed into the Escherichia coli BL21-Codon Plus (DE3)-RIL strains (Stratagene). The positive clones were induced by 0.1 mM isopropyl 1-thio-b-D-galactopyranoside in Luria-Bertani medium at 25 C for 16 h. GST fusion proteins were purified using glutathione Sepharose 4B (GE Healthcare). Purified proteins were desalted by Amicon Ultra-15 centrifugal filter units (Millipore) and stored at À80 C.
The protein samples were separated by SDS-PAGE followed by transfer to polyvinylidene fluoride membranes and western blotting. The following antibodies were used as the primary antibodies in this study: anti-HA antibodies (1:1000 dilution, Roche), anti-c-Myc antibody (1:1000 dilution, Roche), and phophos-(Ser) specific antibodies (1:2000 dilution, CST).
The protein samples were separated by SDS-PAGE followed by transfer to polyvinylidene fluoride membranes and western blotting. The following antibodies were used as the primary antibodies in this study: anti-HA antibodies (1:1000 dilution, Roche), anti-c-Myc antibody (1:1000 dilution, Roche), and phophos-(Ser) specific antibodies (1:2000 dilution, CST).
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