Log In Sign up
The largest database of trusted experimental protocols

Horseradish peroxidase coupled secondary antibody

Manufactured by Santa Cruz Biotechnology
Visit Supplier
Sourced in United States

Horseradish peroxidase-coupled secondary antibodies are a type of laboratory reagent used in various immunoassays and detection methods. They consist of a secondary antibody conjugated to the enzyme horseradish peroxidase (HRP). The HRP enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and visualization of target proteins or molecules in a sample.

Automatically generated - may contain errors

Lab products found in correlation

Tissuelyser 2, by Qiagen (2 mentions) Enhanced chemiluminescence reagent, by PerkinElmer (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Nitrocellulose membrane, by Merck Group (1 mentions) Specific antibodies, by Santa Cruz Biotechnology (1 mentions) Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions) Sc 5579, by Santa Cruz Biotechnology (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Calcein, by Thermo Fisher Scientific (1 mentions) Cox 2 antibody, by Cayman Chemical (1 mentions) Protease inhibitor cocktail set 3, by Merck Group (1 mentions) Ab 9485, by Santa Cruz Biotechnology (1 mentions) Enhanced chemi luminescence (ecl), by Merck Group (1 mentions) Hsp90, by Santa Cruz Biotechnology (1 mentions) Ripa buffer, by Thermo Fisher Scientific (1 mentions) Ecl detection reagent, by Thermo Fisher Scientific (1 mentions) 0.22 μm pvdf membrane, by Roche (1 mentions) Sds sample loading buffer, by Merck Group (1 mentions) Phosphatase and proteinase inhibitors, by Roche (1 mentions)

Spelling variants of this product

Horseradish peroxidase (230 citations) Peroxidase-coupled secondary antibody (16 citations) Horseradish peroxidase (HRP)-coupled secondary antibody (13 citations) Secondary antibody coupled to horseradish peroxidase (2 citations) Horseradish peroxidase-coupled goat anti-rabbit secondary antibody (2 citations)

29 protocols using horseradish peroxidase coupled secondary antibody

1

Western Blot Protocol for Protein Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols
Proteins were separated by electrophoresis through 10% SDS-PAGE and electrophoretically transferred to nitrocellulose membrane (Millipore). After blocking with 5% dried milk in Tris-HCl-buffered saline/0.05% Tween (TBST) for 1 h, membranes were incubated with primary antibodies overnight, washed in TBST, and incubated for 1 h with horseradish peroxidase–coupled secondary antibodies (Santa Cruz). Proteins were detected by using the enhanced chemiluminescence (ECL) systems (Pierce) and Syngene Imaging System.
Zhao M., Joy J., Zhou W., De S., Wood WH I.I.I., Becker K.G., Ji H, & Sen R. (2018). Transcriptional outcomes and kinetic patterning of gene expression in response to NF-κB activation. PLoS Biology, 16(9), e2006347.
+ Open protocol
+ Expand
2

Quantifying Notch 1 Protein Levels

Check if the same lab product or an alternative is used in the 5 most similar protocols
Notch 1, the target gene of miR-146, was detected by western blot analysis following overexpression of miR-146 and shRNA. Cells were lysed using M-PER Protein Extraction Reagent (Pierce) supplemented with a protease inhibitor cocktail (PMSF). Protein concentrations of the extracts were measured using the BCA assays (Pierce) and equalized with extraction reagent. Equal amounts of extracts were loaded and subjected to SDS/PAGE, followed by transfer onto nitrocellulose membranes. Specific antibodies and horseradish peroxidase-coupled secondary antibodies were purchased from Santa Cruz. Membranes were probed using ultra-ECL western blotting detection reagents. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Xiao W.Z., Lu A.Q., Liu X.W., Li Z., Zi Y, & Wang Z.W. (2015). Role of miRNA-146 in proliferation and differentiation of mouse neural stem cells. Bioscience Reports, 35(5), e00245.
+ Open protocol
+ Expand
3

Quantifying AHR Protein Levels

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were lysed on ice in RIPA buffer containing 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Igepal® and 0.25% sodium deoxycholate, with protease inhibitor cocktail (Calbiochem, San Diego, CA, USA). Equal amounts of proteins were applied to SDS-PAGE, transferred onto nitrocellulose membranes and immunoblotted according to the manufacturer’s instructions. The primary antibody against AHR was used at 1:200 (sc-5579; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibody probed blots were visualised with appropriate horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) using enhanced chemiluminescence (34078; Thermo Scientific, Waltham, MA, USA) for detection.
Tkachenko A., Henkler F., Brinkmann J., Sowada J., Genkinger D., Kern C., Tralau T, & Luch A. (2016). The Q-rich/PST domain of the AHR regulates both ligand-induced nuclear transport and nucleocytoplasmic shuttling. Scientific Reports, 6, 32009.
+ Open protocol
+ Expand
4

Evaluating HSP90 Inhibitors in LPS-Activated Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
All reagents were high grade and were purchased from Sigma with the following exceptions. RPMI, DMEM, Calcein and other culture reagents were purchased from Invitrogen Inc (Grand Island, NY, USA) and the UCSF cell culture facility (UCSF, San Francisco, CA). Fetal bovine Serum Defined (FBS) was purchased from Hyclone Laboratories (Logan, UT, USA). Selective HSP90 inhibitors: Geldanamycin, 17-AAG, and radicicol, and BIIB021 were purchased from Calbiochem (San Diego, CA). Lipopolysaccharide (LPS, Escherichia coli, O26:B6) was purchased from Sigma (St Louis, MO). Drugs were dissolved in DMSO or ethanol and stored at −20°C and either used (final concentration of vehicle 0.1% (v/v or dried down and resuspended in PBS/0.1% bovine serum albumin (BSA). HSP antibodies were purchased from Assay design (Enzo Life sciences), for MAP stress activated kinase anti–phospho-JNK/SAPK mAb (#4668) were from Cell Signaling Technology (Danvers, MA); anti–NF-kBp65 (# SC-8008), anti–IkBα (# SC-1643) and respective horseradish peroxidase–coupled secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA) and antibodies against mouse HSP70i (#SPA-810) from Enzo Life sciences, iNOS (# 61043), was from BD Biosciences (BD Biosciences, Lexington, KY); COX-2 antibody is from Cayman Chemical Company (Ann Arbor, MI (# 160106).
Kacimi R, & Yenari M.A. (2015). Pharmacologic heat shock protein 70 induction confers cytoprotection against inflammation in glio-vascular cells. Glia, 63(7), 1200-1212.
+ Open protocol
+ Expand
5

Western Blot Analysis of Skin Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Expression of selected proteins in skin models was verified using Western blotting. For protein extraction skin tissues in PBS (250 µl) were lysed with a TissueLyser II (Qiagen, Hilden, Germany) operated for 5 min at 20 Hz in in presence of Protease Inhibitor Cocktail Set III (3 µl, Merck, Darmstadt, Germany). Extracts equivalent to 30 µg of total protein were then subjected SDS-PAGE and transferred to nitrocellulose membranes following standard protocols. Primary antibodies against TREM-1 (sc-293450), DAP12 (sc-166084) and GAPDH (ab-9485) were used (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Abcam, Cambridge, Great Britain) for subsequent immunostaining, followed by visualisation with appropriate horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) and enhanced chemo-luminescence (34078; Thermo Scientific, Waltham, MA, USA) for detection.
Lemoine L., Dieckmann R., Al Dahouk S., Vincze S., Luch A, & Tralau T. (2020). Microbially competent 3D skin: a test system that reveals insight into host–microbe interactions and their potential toxicological impact. Archives of Toxicology, 94(10), 3487-3502.
+ Open protocol
+ Expand
6

Rac1 Glucosylation Inhibition Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were pre-incubated with the respective inhibitors and treated with TcdB as described above. After 2 h, cells were washed and lysed by freezing. Cell lysates here harvested in 2.5 Laemmli + DTT. Samples were subjected to SDS-PAGE followed by Western blotting. Non-glucosylated Rac1 (1:1000, #610651, BD Biosciences, Franklin Lakes, NJ, USA) as well as Hsp90 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany) were detected by specific primary antibodies and horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) using the enhanced chemiluminescence system (ECL, Millipore, Merck, Darmstadt, Germany). Densitometric quantification of Western blot signals was determined using ImageJ (v1.53p, U.S. National Institutes of Health, Bethesda, MD, USA). Values were normalized to loading control signals (Hsp90).
Braune-Yan M., Jia J., Wahba M., Schmid J., Papatheodorou P., Barth H, & Ernst K. (2023). Domperidone Protects Cells from Intoxication with Clostridioides difficile Toxins by Inhibiting Hsp70-Assisted Membrane Translocation. Toxins, 15(6), 384.
+ Open protocol
+ Expand
7

Western Blot Analysis of hBMSC and OA-CH

Check if the same lab product or an alternative is used in the 5 most similar protocols
hBMSC and OA-CH were washed two times with PBS and lysed with RIPA buffer (Thermo Scientific, Waltham, MA) containing phosphatase and proteinase inhibitors (Roche, Germany). The concentration of cellular protein and EVs was quantified using a BCA protein kit assay (see hBMSC EV Isolation). Cell lysates (10 ug) or EVs (10 ug) were mixed with SDS-sample loading buffer (#B7053, Sigma-Aldrich, Germany), followed by boiling for 5 min at 95°C, and subjected to 12% SDS-PAGE. The proteins were transferred to 0.22 μm PVDF membranes (Roche, Penzberg, Germany) after electrophoretic separation. Blot membranes were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibodies on a shaker overnight at 4°C. After washing, the membranes were incubated with the appropriate horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology and Jackson ImmunoResearch, West Grove, PA). Proteins were examined using ECL detection reagents (Thermo Scientific; Germany), and signals from cell lysates were normalized to β-actin (except for EV band analysis). Antibodies used for this study are listed in Table 2.
Li S., Stöckl S., Lukas C., Götz J., Herrmann M., Federlin M, & Grässel S. (2020). hBMSC-Derived Extracellular Vesicles Attenuate IL-1β-Induced Catabolic Effects on OA-Chondrocytes by Regulating Pro-inflammatory Signaling Pathways. Frontiers in Bioengineering and Biotechnology, 8, 603598.
+ Open protocol
+ Expand
8

Immunoblotting Analysis of GDNF Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols
Briefly, cells were homogenized in lysis buffer (25 mM Tris–HCl pH 7, 1% IGEPAL, 100 mM NaCl) and supplemented with protease inhibitors (Roche). Proteins were obtained by centrifugation at 13,800 g at 4°C for 15 min, and quantified by Bradford assay (Bio-Rad). Proteins (30 μg) were resolved on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Bioscience). The membranes were blocked with 5% non-fat milk and incubated overnight with primary antibodies. Pre-stained markers (Invitrogen) were included for size determination. The following antibodies were used: rabbit anti-GDNF (1:1000, sc-9010 Santa Cruz Biotechnology) and anti-GAPDH (1:1000, MAB374 Millipore). Membranes were washed and incubated with corresponding horseradish peroxidase-coupled secondary antibodies (1:10,000, Santa Cruz) for 2 h. Immunoreactive bands were detected using the enhanced chemiluminescence method (Amersham Bioscience) and developed on photographic film (Kodak).
Cortés D., Robledo-Arratia Y., Hernández-Martínez R., Escobedo-Ávila I., Bargas J, & Velasco I. (2016). Transgenic GDNF Positively Influences Proliferation, Differentiation, Maturation and Survival of Motor Neurons Produced from Mouse Embryonic Stem Cells. Frontiers in Cellular Neuroscience, 10, 217.
+ Open protocol
+ Expand
9

Western Blotting of TSPO Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols
Western blotting was performed to confirm expression of stably over-expressing TSPO WT or A147T using previously described methods.44 (link) Primary antibodies were to human TSPO (Abcam); V5 (Life Technologies) and GAPDH (Millipore). Horseradish peroxidase-coupled secondary antibodies (Santa Cruz) were used. Western blots were detected on a ChemiDoc imaging system (BioRad) and quantified using Fiji software.45 (link)
Sokias R., Werry E.L., Chua S.W., Reekie T.A., Munoz L., Wong E.C., Ittner L.M, & Kassiou M. (2016). Determination and reduction of translocator protein (TSPO) ligand rs6971 discrimination †The authors declare no competing interests.. MedChemComm, 8(1), 202-210.
+ Open protocol
+ Expand
10

Western Blot Protein Analysis Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Western blotting analyses were performed as previously described (7 , 9 (link), 10 (link), 27 (link)). For Western blotting experiments, 30 μg of total protein was loaded and proteins were separated by SDS-PAGE (200 V for 40 min) and electrophoretically transferred to a nitrocellulose filters (semi-dry transfer at 10 V for 30 min). Filters were then blocked with 5% non-fat dry milk in Tris buffered saline (20 mM Tris, pH 7.6, 137 mM NaCl) with 0.1% Tween 20, washed, and then incubated with primary antibody. The blots were incubated with antibodies against rabbit polyclonal p47phox and p67phox, (Santa Cruz Biotechnology Inc., CA, USA) and beta-actin (Cell signaling). After incubation with the primary antibody, the bound antibody was visualized with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The expression levels of each protein were quantified by densitometric analysis of corresponding band using Scion image software (28 (link)).
Thandavarayan R.A., Garikipati V.N., Joladarashi D., Babu S.S., Jeyabal P., Verma S.K., Mackie A.R., Khan M., Arumugam S., Watanabe K., Kishore R, & Krishnamurthy P. (2015). Sirtuin-6 deficiency exacerbates diabetes induced impairment of wound healing. Experimental dermatology, 24(10), 773-778.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!