Cell extracts were prepared from a 1 mL aliquot of S. venezuelae cells grown in liquid MYM medium. The protein extracts were separated using 15% SDS-PAGE and were stained with Coomassie brilliant blue R-250. Equivalent amounts of total protein were loaded onto a second 15% SDS-PAG, and following transfer to PVDF membranes, were subjected to immunoblotting with anti-FLAG antibodies (1:1,500; Sigma) and anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000; Cell Signaling).
Horseradish peroxidase hrp conjugated anti rabbit igg secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody is a lab equipment product. It is a secondary antibody that targets rabbit IgG and is conjugated with the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti flag antibody, by Merck Group (1 mentions)
T p38 mapk, by Cell Signaling Technology (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
P iκb, by Cell Signaling Technology (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Camptothecin, by Merck Group (1 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
T p65 nfκb, by Cell Signaling Technology (1 mentions)
Synergy mx microplate reader, by Agilent Technologies (1 mentions)
T iκb, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Luminescence reagent set, by Fujifilm (1 mentions)
Rpmi 1640 medium, by Fujifilm (1 mentions)
Gapdh, by Fujifilm (1 mentions)
Phosphorylated erk1 2 p erk1 2, by Cell Signaling Technology (1 mentions)
8 protocols using horseradish peroxidase hrp conjugated anti rabbit igg secondary antibody
1
Immunoblotting of S. venezuelae Proteins
2
Inflammatory Cytokine Regulation Assay
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Streptomycin (100 ng/mL) and penicillin (100 U/mL) were purchased from Sigma (Gillingham, UK); 96 well plates were provided by NUNC, (Havel, Germany), while GIBCO (Grand island, NY) provided fetal bovine serum (FBS). DMSO, dexamethasone, lipopolysaccharides (LPS—Escherichia coli O111:B4), N-nitro-l -arginine methyl ester (L-NAME), Griess reagent (1% sulfanilamide/0.1% naphthyl ethylenediamine dihydrochloride in 2.5% H3PO4), camptothecin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma, IL-6 and TNF-α ELISA Kits were procured from Invitrogen (Massachusetts, US). Synergy Mx microplate reader belonged to BioTek, Winooski, VT, USA. P-IκB, T-IκB, P-p65-NF-κB, T-p65-NF-κB, P-p38-MAPK, and T-p38-MAPK, GAPDH, and anti-rabbit IgG horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
3
Quantitative Western Blot Analysis
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Western blotting was performed using a standard protocol.76 The following primary antibodies were used at a dilution of 1:1,000: BRCA1 (D54A8, #9025), BRCA2 rabbit (H‐300, sc‐8326, #9012), CD44 mouse (8E2, #5640), PTEN (D4.3, #9188), EGFR rabbit, ß‐actin mouse, and ß‐actin rabbit (13E5, #4979; all Cell Signaling Technology, Danvers, MA). Anti‐rabbit IgG horseradish peroxidase (HRP)‐conjugated secondary antibody (#7074; Cell Signaling Technology) was used. Protein expression was visualized with ECL prime western blotting detection reagent substrate on an Image Quant LAS4000 imager.
4
D-Leu Modulates Stress Responses in K562 Cells
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Human K562 leukemia cells were cultured in RPMI‐1640 medium (Fujifilm Wako Pure Chemical Co.) supplemented with 3% fetal bovine serum, penicillin, and streptomycin for 48 h, followed by incubation with various concentration of d ‐Leu (0‐10 μmol/L) for 24 h at 37°C in 5% CO2. The cells were collected analyzed by western blotting. Membranes were immunostained with primary antibodies against Nrt2 (1:2000 dilution, Cusabio Technology LLC.), HO‐1 (1:8000 dilution, Gene Tex Inc), SOD2 (1:4000 dilution, Cusabio LLC.), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 1:8000 dilution, Fujifilm Wako Pure Chemical Co.), followed by incubation with anti‐rabbit IgG horseradish peroxidase (HRP)‐conjugated secondary antibody (Cell Signaling Technology Inc) for 1 hour at room temperature. Immunoreactive bands were visualized using a Luminescence Reagent Set (Wako Pure Chemical Industries, Ltd) and detected with Image Quant LAS500 (GE Healthcare Japan Com., Tokyo, Japan). The intensities of the detected bands were calculated using ImageJ software.
5
Reagents and Antibodies Used in Cellular Assays
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The reagents used in the present study were purchased from the following suppliers: FR and API-1 from Tocris Bioscience (Bristol, UK); RPMI-1640 medium, fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA); water soluble tetrazolium-1 (WST-1), Cytotoxicity Detection Kit Plus, Cell Proliferation ELISA colorimetric kit and Cell Death Detection ELISA Plus kit from Roche Diagnostics GmbH (Mannheim, Germany); and PathScan ® Cleaved Caspase-3 (Asp175) Sandwich ELISA kit and monoclonal rabbit antibodies against β-actin (ACTB; catalog no., 4970; dilution, 1:1,000), B-cell lymphoma-2 (BCL2)-associated X protein (BAX; catalog no., 5023; dilution, 1:1,000), BCL2 antagonist/killer (BAK; catalog no., 12105; dilution, 1:1,000), cyclin D1 (CYCD1; catalog no., 2978; dilution, 1:1,000), cMYC (catalog no., 13987; dilution, 1:1,000), Akt (catalog no., 4685; dilution, 1:1,000), ERK1/2 (catalog no., 4370; 1:2,000), phosphorylated Akt (pAkt; catalog no., 4060; dilution, 1:2,000), phosphorylated ERK1/2 (pERK1/2; catalog no., 4094; dilution, 1:1,000) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (catalog no., 7074; dilution, 1,1000) were provided by Cell Signaling Technology (Danvers, MA, USA). All other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
6
Exosome Protein Profiling by Western Blot
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Exosome preparations, normalized to 20 μg protein content, were mixed with 5× Laemmli sample buffer (Elpisbio, Daejeon, Korea) and boiled for 5 min at 95 °C. Exosome proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4%–20% gradient polyacrylamide gels (Cat# 456–1093; Bio-Rad, CA, USA) and subsequently transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk solution in 1× Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) for 1 h, the membrane was incubated overnight at 4 °C with anti-CD63 (1:1000, bs-1523; Bioss, MA, USA), anti-Alix (1:1000, ab88388; Abcam, Cambridge, UK), anti-CD81 (1:1000, bs-6943R; Bioss, MA, USA) and anti-Annexin V (1:1000, SC-8300; Santa Cruz Biotechnology, CA, USA) primary antibodies. The membrane was washed 5 times in 1× TBST for 1 h at room temperature (RT) and then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (1:2000 in TBST containing 5% skim milk; Cell Signaling Technology, MA, USA). After washing five times in 1× TBST for 1 h at room temperature, immunoreactive bands were detected using the Clarity Western Enhanced Chemiluminescence (ECL) kit (Bio-Rad, Cat #: 170–500) and Chemidoc imaging system (Bio-Rad).
7
Biochemical and Immunological Assays for Neurodegeneration
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Dulbecco’s Modified Eagle’s Medium (DMEM), calf serum (CS), penicillin, streptomycin, Dimethyl sulfoxide (DMSO), MTT, and DCF-DA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). A SOD kit was obtained from Dojindo Molecular Technologies (Rockville, MD, USA). Ethanol was purchased from Ethanol Supplies World Co., Ltd. (Jeonju, Republic of Korea). Dimethyl sulfoxide (DMSO), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), o-phthaldialdehyde (OPT), acetylthiocholine, AChE, pyruvic acid, malic acid, mannitol, egtazic acid (EGTA), hydroxyethyl piperazine ethane sulfonic acid (HEPES), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1), N,O-bis(trimethylsilyl)trifluoroacetamide(BSTFA), and all other reagents used were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
The primary and secondary antibodies are listed below: ACh, AChE, SYP, PSD-95, GAP-43, TLR-4, NF-κB, IκB-α, IL-1β, TNF-α, BCL-2, BAX, caspase-3, ZO-1, occludin, claudin-1, and β-actin were purchased from Santa Cruz Biotech (Dallas, TX, USA). ChAT and Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody were purchased from Cell Signaling Tech (Danvers, MA, USA). HRP-conjugated anti-mouse IgG secondary antibody purchased from Bio-Rad (Hercules, CA, USA).
The primary and secondary antibodies are listed below: ACh, AChE, SYP, PSD-95, GAP-43, TLR-4, NF-κB, IκB-α, IL-1β, TNF-α, BCL-2, BAX, caspase-3, ZO-1, occludin, claudin-1, and β-actin were purchased from Santa Cruz Biotech (Dallas, TX, USA). ChAT and Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody were purchased from Cell Signaling Tech (Danvers, MA, USA). HRP-conjugated anti-mouse IgG secondary antibody purchased from Bio-Rad (Hercules, CA, USA).
8
Western Blot Analysis of Protein Lysates
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Cells were harvested by trypsinization, and lysed with Pink Buffer as previously described14 (link). Ten micrograms of lysate was resolved on NuPAGE 4-12% Bis-Tris gels (Life Technologies) and electrophoretically transferred to a nitrocellulose membrane using iBlot Dry Blotting System (Life Technologies). Membranes were blocked for 1 hr in 5% BSA in Tris-buffered saline with Tween 20 (TBST, Dako), and then incubated overnight with specific primary antibodies in 5% BSA in TBST. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (1:2000, Cell Signaling Technologies) was diluted in 5% nonfat dry milk in TBST. Chemiluminescence detection reagents were incubated with the membrane, and an image was acquired using an Image Quant LAS500 (GE Healthcare). Band intensities were quantified using ImageJ version 1.49q ( http://rsbweb.nih.gov/ij/ ). Primary antibodies used in this analysis are shown in Supplementary Table S2 .
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