Aliquots of 50 μl of A600-normalized overnight cultures grown at 37 °C were mixed with 50 μl of 6× SDS-loading buffer and resolved in 20% Tricine-SDS gels (96 (link)). Assessment of the RhaPS production was performed via immunoblotting on polyvinylidene difluoride membranes following the traditional immunoblotting technique. Primary antibody was rabbit-raised anti-S. pyogenes Group A carbohydrate polyclonal antibody (Abcam, ab21034). Secondary antibody was goat-raised anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, 170-6515). Immunoreactive signals were captured using either GENESYSTM 10S UV-visible spectrophotometer (Thermo Scientific) after exposure to the Clarity Western ECL (Bio-Rad).
Anti rabbit igg horseradish peroxidase conjugate
Manufactured by Bio-Rad
Sourced in United States
Anti-rabbit IgG horseradish peroxidase conjugate is a labeling reagent used in various immunoassay techniques. It consists of horseradish peroxidase enzyme conjugated to anti-rabbit immunoglobulin G (IgG) antibodies. This conjugate can be used to detect and quantify the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl, by Bio-Rad (1 mentions)
Amicon ultra 10k, by Merck Group (1 mentions)
Anti hsp70, by Abcam (1 mentions)
Clarity western ecl substrate chemiluminescence kit, by Bio-Rad (1 mentions)
Anti cd63, by Abcam (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Forskolin, by Merck Group (1 mentions)
Xt mes electrophoresis buffer, by Bio-Rad (1 mentions)
Insulin actrapid, by Novo Nordisk (1 mentions)
Goat anti mouse igg horseradish peroxidase conjugate, by Bio-Rad (1 mentions)
Taq man universal master mix with ung, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Dibutyryl camp db camp, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
E z n a hp total rna kit, by Omega Bio-Tek (1 mentions)
4 protocols using anti rabbit igg horseradish peroxidase conjugate
1
Quantifying RhaPS Expression via Immunoblot
2
Immunoblotting for AtNUDX9 Protein
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Western blot analysis was carried out as described previously by Yoshimura et al. (2004) (link). Protein bands were detected by an anti-AtNUDX9 polyclonal rabbit antibody prepared using the recombinant protein as the primary antibody and an anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad) as the secondary antibody.
3
Extracellular Vesicle Protein Analysis
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The SEC fractions were concentrated with Amicon ® Ultra 10K (Merck) concentrator. The total protein concentration of samples was measured with a DC protein assay (Bio-Rad, Hercules, CA, USA). On each lane of the 12% m/v SDS-PAGE gel, 30 mg of total protein amount from certain fractions was loaded. The proteins were separated at 100V for 90 min. Next, the proteins were blotted onto a PVDF membrane by using a wet transfer system at 100 V for 1 h. The membrane was blocked with TBST buffer with 3% m/v skim milk for 1 h at 4°C. The proteins were detected using the primary rabbit monoclonal antibodies: anti-Hsp70, anti-TSG101, anti-CD63 (Abcam, Trumpington, UK), and the secondary antibodies anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA). The signals were measured in the ChemiDoc Touch Imaging system (Bio-Rad, USA) with a Clarity Western ECL Substrate chemiluminescence kit (Bio-Rad, USA).
4
Comprehensive Cell Culture Reagent Catalog
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Cell culture flasks and plates were purchased from Sarstedt (Nümbrecht, Germany) or TPP (Transandigrn, Switzerland). Advanced MEM, DMEM, OptiMEM, GlutaMAX, MEM vitamin solution, fetal bovine serum (FBS), Fungizone (250 µg/mL of amphotericin B), gentamicin (10 mg/mL), trypsin-EDTA, Pierce Enhanced Chemiluminescence (ECL) Western Blotting substrate, High Capacity cDNA Reverse Transcription Kit, TaqMan Universal Master Mix with UNG and TaqMan gene expression assays were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.). 4-12% Criterion Bis-Tris polyacrylamide gels, XT MES electrophoresis buffer, goat anti-mouse IgG-horseradish peroxidase conjugate, anti-rabbit IgG-horseradish peroxidase conjugate, PCR plates and PCR plate sealing films were purchased from Bio-Rad (Hercules, CA, U.S.). Amersham ECL Full-Range Rainbow Molecular Weight Marker was purchased from (Cytiva, U.S.). Polyvinylidene fluoride (PVDF) membrane, forskolin, dibutyryl-cAMP (db-cAMP), bovine serum albumin (BSA), and all other reagents were purchased from Merck/Sigma-Aldrich (Darmstadt, Germany). Insulin (Actrapid) was obtained from NovoNordisk (Denmark). E.Z.N.A HP Total RNA Kit was purchased from Omega Bio-Tek (Norcross, GA, U.S.). Dexamethasone was purchased from KRKA (Slovenia).
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