Cell culture media (RPMI, DMEM, DMEM/F12 (1:1),), fetal bovine serum (FBS), antibiotics (streptomycin, penicillin), L-glutamine, and 5-aminolevulinic acid (ALA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cayman Chemicals (Ann Arbor, MI, USA) supplied the following reagents: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-[3 (aminomethyl) benzyl] acetamidine (1400W), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolone-1-oxyl-3-oxide (cPTIO), DETA-NONOate (DETA/NO), and a polyclonal antibody against human iNOS. A rabbit monoclonal antibody against human β-actin and horseradish peroxidase-conjugated IgG secondary antibodies were from Cell Signaling Technologies (Danvers, MA). Pierce chemical Co. (Rockford, IL) supplied reagents for bicinchronic (BCA) protein assays and for SuperSignal West Pico chemiluminescence detection of proteins on Western blots. The silicone flexi-Perm® micro-12 inserts and flexi-Perm-ConA® rings for separating targeted cells from bystanders were obtained from Sarstedt-AG (Numbrecht, Germany).
Horseradish peroxidase conjugated igg secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated IgG secondary antibodies are laboratory reagents used in various immunoassays and detection techniques. They consist of secondary antibodies (typically raised against the primary antibody's species) that are conjugated to the enzyme horseradish peroxidase. This enzyme-antibody conjugate can be used to detect and amplify the signal from primary antibodies that have bound to their target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Increased chemiluminescence, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Phosphate buffered saline (pbs), by Welgene (2 mentions)
5 aminolevulinic acid ala, by Merck Group (1 mentions)
Human β actin, by Cell Signaling Technology (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Srebp 1c, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by Abcam (1 mentions)
Interleukin 6 (il 6), by Novus Biologicals (1 mentions)
Caspase 1, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
14 protocols using horseradish peroxidase conjugated igg secondary antibody
1
Cell Culture Materials and Reagents
2
Western Blot Analysis of Autophagy and Inflammation Markers in Liver Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver samples were homogenized and centrifuged using RIPA lysis buffer (Millipore, Bedford, MA, USA) containing a protease inhibitor cocktail with 1 mmol/L phenylmethanesulfonyl fluoride. Protein lysates were quantified using the BCA kit (Thermo Scientific, Waltham, MA, USA). Twenty micrograms of protein extract were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred to a nitrocellulose membrane. After blocking in 5% bovine serum albumin for 1 h, membranes were then incubated overnight at 4 °C with the following primary antibodies: LC3, ATG7, p62/SQSTM1, mTOR, p-mTOR, ULK, Beclin1 ATG5-12, SOD2, β-actin, NLRP3, ASC, caspase 1, HSL, ATGL, and FAS (all 1 : 2000, Cell Signaling Technology, Danvers, MA, USA); SREBP-1c (1 : 2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA); IL-6 (1:1,000, Novus Biologicals, Centennial, CO, USA); HO-1 ( 1: 2000, Enzo Life Sciences, Farmingdale, NY, USA); and TNF-α (1 : 1000, Abcam, Cambridge, MA, USA) at 4 °C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated IgG secondary antibodies (1:3000, Cell Signaling). The density of the individual protein bands was quantified by densitometric scanning of the blots using ImageJ software (version 1.50d, USA). Expression levels were then normalized to the ctrl group, which were set to 1.
3
Quantification of Apoptosis Regulators in Thalamus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iated and mature brain astrocytes The remaining six rats in each group were killed to measure the levels of Bcl-2 and Bax proteins in the thalamus by Western blot analysis. Under deep anesthesia, the rats were perfused intracardially with 50 ml ice-cold 0.9% saline. The right thalamus was immediately isolated on ice and homogenized in cell lysis buffer. The protein samples were separated on a 12% sodium dodecyl sulfate (SDS)/polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, United States). The membranes were incubated with Bcl-2, Bax, and glyceraldehyde phosphate dehydrogenase (GAPDH) antibodies (Cell Signaling Technology, United States) at 4°C overnight. Immunoreactive bands were visualized by increased chemiluminescence (Millipore, United States) using corresponding horseradish peroxidase-conjugated IgG secondary antibodies (Cell Signaling Technology, United States). The images were captured by the gel imager and quantified using Quantity One software (Bio-Rad, United States).
4
Protein Expression Analysis in Rat Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of proteins extracted from the primary neurons and the right cerebral cortex of rats was analysed by Western blot. The protein samples were separated on a 12% sodium dodecyl sulfate (SDS)/polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated with GLP-1R (Abcom, UK), phosphorylated AKT, ERK, p38, JNK and total AKT, ERK, p38, JNK, Bcl-2, Bax, Bcl-xl, Bad, cleaved caspase-3, -8, and -9, cleaved PRAP and GAPDH antibodies (Cell Signaling Technology, USA) at 4 °C overnight. Immunoreactive bands were visualized by increased chemiluminescence (Millipore, USA) using corresponding horseradish peroxidase-conjugated IgG secondary antibodies (Cell Signaling Technology, USA). The images were captured by the gel imager (UVITEC, UK) and quantified using Quantity One software (Bio-Rad, USA).
5
Protein Expression Analysis of Differentiated hNSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from the differentiated hNSCs using RIPA lysis buffer with 1 mM phenylmethanesulfonylfluoride. A total of 20 μg of protein samples was separated by a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes. Each membrane was blocked for 1 h with 5% defatted milk powder at room temperature and then incubated with TUBB3 (1:1000, #ab53623, Abcam, Cambridge, UK), ATG5 (1:1000, #12994, Cell Signaling Technology, Danvers, MA, USA), BECN1 (1:1000, #3495, Cell Signaling Technology, Danvers, MA, USA), LC3A/B (1:1000, #12741, Cell Signaling Technology, Danvers, MA, USA), CTNNB (1:1000, #8480, Cell Signaling Technology, Danvers, MA, USA), MYC (1:1000, #5605, Cell Signaling Technology, Danvers, MA, USA) or ACTB (1:5000, #AB2001, ABways, Shanghai, China) primary antibody at 4 °C overnight. The membrane was then incubated with corresponding horseradish peroxidase-conjugated IgG secondary antibodies (1:10,000, #7074P2, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The blot images were captured by the gel Bio-rad imager. The gray value of each band was measured using ImageJ. The relative expression of each band was calculated by comparing the target protein band with GAPDH. Each group had three independent samples.
6
Antioxidant and Neuroprotective Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco’s Modified Eagle Medium (DMEM), 1× PBS and 1× Tris-buffered saline (TBS) were purchased from Welgene, Inc. (Gyeongsan, Gyeongbuk, Korea). Fetal bovine serum (FBS), 0.25% trypsin-EDTA, antibiotics, and 2,7-dichlorofluorescein diacetate (DCFDA) were obtained from Invitrogen (Carlsbad, CA, USA). K252a (a TrkB inhibitor) and MK-2206 (a specific Akt inhibitor) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). EZ-Cytox cell viability assay kit was obtained from Daeil Lab (Seoul, Korea). Specific antibodies against heme-oxygenase-1(HO-1), glutamate–cysteine ligase catalytic subunit (GCLC), and NAD(P)H quinone oxidoreductase-1 (NQO-1), NF-E2-related factor-2 (Nrf2), phosphoprotein kinase B(Akt), extracellular signal-regulated kinase (ERK), phospho-cAMP response element-binding protein (CREB), phospho-tropomyosin-related kinase receptor B(TrkB), and β-actin as well as horseradish peroxidase-conjugated IgG secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Specific antibodies against BDNF (sc-65514) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Muse® Annexin V & Dead Cell Assay Kits were purchased from Merck Millipore, Inc. (Darmstadt, Germany). All other chemicals used in this study were analytical grade and procured from Sigma-Aldrich (St Louis, MO, USA).
7
Protein Expression Analysis in Rat Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expressions of proteins extracted from the primary astrocytes and the right cerebral cortex of rats were analyzed by Western blot. Proteins in the samples were separated on a 12% sodium dodecyl sulfate (SDS)/polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated with the primary antibodies including anti-caspase-1 (1:500, ab108362, Abcam Cambridge, UK); anti-NLRP3 (1:1, 000; Cryo-2, AdipoGen); anti-ASC (N-15) (1:1, 000; Santa Cruz), anti-HMGB-1 (1:500, ab79823, Abcam Cambridge, UK), anti-RAGE (1:500, ab181293, Abcam Cambridge, UK), PPAPɤ (1: 1000, ab41928, Abcam Cambridge, UK), GAPDH (1:5000, #5014, Cell Signaling Technology, USA) and Histone H3 (1:1000, #12648, Abcam Cambridge, UK, loading control for nuclear proteins) at 4 °C overnight. Immuno-reactive bands were visualized by increased chemiluminescence (Millipore, USA) using corresponding horseradish peroxidase-conjugated IgG secondary antibodies (Cell Signaling Technology, USA). The images were captured with the gel imager (UVITEC, UK) and quantified using Quantity One software (Bio-Rad, USA).
8
Quantitative Western Blot Analysis of Apoptosis-Related Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from HLE-B3 cells using RIPA lysis buffer supplemented with a protein inhibitor cocktail (Roche Applied Science). Protein concentration determination was carried out using a BCA kit (Thermo Fisher Scientific, Inc.). Protein samples (15 µg per lane) were separated via 8% SDS-PAGE and the separated proteins were transferred onto PVDF membranes. The PVDF membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated with anti-BCL2 (cat. no. 4223; 1:1,000; Cell Signaling Technology, Inc.), anti-caspase-3 (cat. no. 14220, 1:1,000; Cell Signaling Technology, Inc.), anti-caspase-8 (cat. no. 4790; 1:1,000; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. 2118; 1:1,000; Cell Signaling Technology, Inc.) primary antibodies overnight at 4˚C. Following the primary antibody incubation, the membranes were incubated with a horseradish peroxidase-conjugated IgG secondary antibody (cat. no. 5127; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. Protein bands were visualized using an ECL chemiluminescence Substrate Reagent kit (Pierce; Thermo Fisher Scientific, Inc.). The densitometry of protein was normalized to GAPDH and analyzed using ImageJ (version 1.5.2; National Institutes of Health).
9
Leydig Cell Cultures for Testosterone Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leydig TM3 cells were obtained from ATCC (UK). Merck supplied the dibutyryl cyclic AMP (Bt2cAMP), Neutral Red, GPX4 inhibitor RSL3, Ebselen, horse serum, FBS, BSA, Hepes, Penicillin/Streptomycin, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Percoll, Collagenase D, Dispase, DNase I, X-TremeGENE transfection reagent, Insulin-Transferrin-Sodium Selenite Supplement (TIS), Complete Mini (a Roche mixture of protease inhibitors) and Dulbecco’s modified Eagle’s/Ham’s nutrient F12 (DME/F12) medium. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol were obtained from Avanti Polar Lipids. ThermoFisher Scientific provided M-199 medium, Opti-MEM medium, Soybean Trypsin Inhibitor (STI), DPBS, HBSS, EGF, C11-BODIPY(581/591), Mitotracker Deep Red, and primary rabbit antibodies against StarD1 and StarD4. A testosterone assay kit and primary rabbit antibodies against GPx4 and β-actin were obtained from Abcam. A horseradish peroxidase-conjugated IgG secondary antibody was from Cell Signaling Technology. Gel electrophoresis consumables were from BioRad. 7-OOH was prepared by dye-sensitized photoperoxidation of Ch, as described [34 (link)]. Stock solutions of 7-OOH in 2-propanol were standardized for hydroperoxide content by iodometric analysis [22 (link)] and stored at −20 °C.
10
Western Blot Analysis of Immune Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As previously described, “total cell lysates were extracted with lysis buffer containing 20 mM HEPES pH 7.5; 1.5 mM KCl; 1 mM EDTA; 1 mM EGTA; 0.15% Triton-X100; 1 mM PMSF; 1 mM DTT; and a cocktail of protease inhibitors (Sigma). The protein content of the lysate was measured using BCA protein assay reagent (Pierce). Aliquots containing an equal amount of protein (30 μg) were separated by SDS-PAGE and then transferred onto PVDF membranes (Sigma). Blots were blocked with 5% non-fat dry milk in PBS Tween 0.1% (PBST) and then incubated with primary antibodies overnight” (30 (link)): anti-phosphoIRF3 antibody (Cell Signaling−4947), anti-phospo-STAT1 (Cell Signaling−9167), anti-STAT1 (Cell Signaling−9172), anti-α-Tubulin (Cell Signaling−2144). Next, blots were incubated with corresponding horseradish peroxidase–conjugated IgG secondary antibody (anti-rabbit or anti-mouse, Cell Signaling). Detection of immunoreactive bands detection was carried out using the enhanced chemiluminescence (ECL) kit (Amersham) according to the manufacturer's instructions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!