Horseradish peroxidase hrp conjugated secondary antibody

Manufactured by Promega

Sourced in United States

Horseradish peroxidase (HRP)-conjugated secondary antibodies are enzyme-labeled antibodies used in various immunodetection techniques. HRP is an enzyme that catalyzes a colorimetric reaction, allowing for the visualization and quantification of target proteins.

Automatically generated - may contain errors

Lab products found in correlation

Protein assay, by Bio-Rad (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Phospho jnk thr183 tyr185, by Cell Signaling Technology (1 mentions) β actin, by Abcam (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Ecl blocking agent, by GE Healthcare (1 mentions) Ecl advanced, by GE Healthcare (1 mentions) Ecl plus, by GE Healthcare (1 mentions) Complete antiprotease cocktail, by Roche (1 mentions) Las4000 imager, by GE Healthcare (1 mentions) Pvdf membrane, by Cytiva (1 mentions) Gradient 4 20 sds page gels, by Bio-Rad (1 mentions) Gapdh, by Merck Group (1 mentions) Mcl 1, by Santa Cruz Biotechnology (1 mentions) Ecl reagent, by Bio-Rad (1 mentions) Pcdk9, by Cell Signaling Technology (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Anti calnexin, by Enzo Life Sciences (1 mentions)

15 protocols using horseradish peroxidase hrp conjugated secondary antibody

Investigating NF-κB and MAPK Signaling in LPS-Induced RAW 264.7 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The effects of FLR and LA on NF-κB and MAPK signaling in LPS-induced RAW 264.7 cells were examined by western blot analysis. The details of the western blot analysis have been described in our previous study [37 (link)]. Antibodies against NF-κB p65, phospho-NF-κB p65 (Ser536), IκBα, phospho-IκBα (Ser32), ERK, phospho-ERK (Thr202/Tyr204), p38, phospho-p38 (Thr180/Tyr182), JNK, phospho-JNK (Thr183/Tyr185) were obtained from Cell Signaling Technology (Danvers, MA, USA), and β-actin was obtained from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Promega (Madison, WI, USA).

Kim S.M., Park E.J., Kim J.Y., Choi J, & Lee H.J. (2020). Anti-Inflammatory Effects of Fermented Lotus Root and Linoleic Acid in Lipopolysaccharide-Induced RAW 264.7 Cells. Life, 10(11), 293.

+ Open protocol

+ Expand

Western Blot Analysis of Cellular Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in radio immunoprecipitation assay (RIPA) [100 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sufate] buffer with a protease inhibitor cocktail (Sigma-Aldrich). The protein concentrations in the lysates were determined by bicinchoninic acid (BCA) assay and a total of 30 μg of protein samples were resolved on 10% SDS-PAGE. After the electrophoretic transfer of proteins onto the nitrocellulose membrane, the membranes were incubated overnight at 4°C with primary antibodies using the following dilutions: EAAT1 (1:1000), YY1 (1:500), NF-κB (1:500), IκBα (1:500), β-actin (1:1000) histone H3 (1:500) and Na⁺/K⁺-ATPase (1:500) phosphor-Akt (1:500) and total Akt (1:1000), phosphor-ERK (1:500) and total ERK (1:1000). Afterwards, incubation was performed with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, Promega) at room temperature for 1 h. The blots were developed and detected with an enhanced chemiluminescence detection kit (Pierce).

Karki P., Hong P., Johnson J J.r., Pajarillo E., Son D.S., Aschner M, & Lee E. (2017). Arundic acid increases expression and function of astrocytic glutamate transporter EAAT1 via the ERK, Akt and NF-κB pathways. Molecular neurobiology, 55(6), 5031-5046.

+ Open protocol

+ Expand

Western Blotting Technique for Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total cell and tissue lysates were prepared as reported previously [33 (link)]. Equal protein samples (typically 5–10 μg protein/lane) were subjected to electrophoresis using 8, 10 or 12% SDS-polyacrylamide gels, depending on the molecular weight of the protein of interest. Proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) by a liquid transfer method. Membranes were blocked with ECL blocking agent (GE Healthcare) in Tris-Buffer saline containing 0.05% Tween-20 (TBS-T) and incubated overnight with a 1:1000 dilution of each primary antibody. The membranes were washed 3 times with TBS-T prior to incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega). Immunoreactive protein bands were detected by enhanced chemiluminescence (ECL) using ECL Advanced or ECL Plus (GE Healthcare) depending on signal intensity. The expression of β-actin was used as a loading and reference control.

Bacallao K, & Monje P.V. (2015). Requirement of cAMP Signaling for Schwann Cell Differentiation Restricts the Onset of Myelination. PLoS ONE, 10(2), e0116948.

+ Open protocol

+ Expand

Western Blot Analysis of Cell Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols

At the end of the treatment, cells were washed twice with ice-cold phosphate buffered saline (PBS) and cell lysates were prepared by adding radioimmunoprecipitation assay (RIPA) buffer with a protease inhibitor cocktail. The protein concentration was determined by BCA assay and 30 μg of total protein were run on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane for western blot analysis. The primary antibodies were used at 1:1000 dilution and horse radish peroxidase (HRP)-conjugated secondary antibodies (Promega) were used at 1:5000 dilution. The blots were developed with enhanced chemiluminescence detection kit (Pierce).

Karki P., Johnson J J.r., Son D.S., Aschner M, & Lee E. (2016). Transcriptional regulation of human transforming growth factor-α in astrocytes. Molecular neurobiology, 54(2), 964-976.

+ Open protocol

+ Expand

Whole-Cell Lysis and Western Blot Assay

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Preparation of whole-cell cell lysates and Western blot analysis were performed as described previously (9 (link)). Briefly, cells were scraped and centrifuged at 4⁰C. Pellets were lysed in RIPA lysis buffer with 1 x Halt protease and phosphatase inhibitors cocktail (sc-364162, Santa Cruz Biotechnology). Protein concentrations were measured using Bio-Rad protein Assay (Bio-Rad). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to nitrocellulose membranes (0.45 µm, 1620115, Bio-Rad). Membranes were probed with specific primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega). Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate detection reagent (P90720, Sigma-Aldrich). Images were taken on the ChemiDoc XRS System (Bio-Rad) and the protein bands intensities were quantified by NIH ImageJ software. The relative values for various proteins regulated by autophagy induced by glucose starvation or metformin were calculated and shown as a ratio relative to control treatments.

Hong J., Maacha S., Pidkovka N., Bates A., Salaria S.N., Washington M.K, & Belkhiri A. (2022). AXL Promotes Metformin-Induced Apoptosis Through Mediation of Autophagy by Activating ROS-AMPK-ULK1 Signaling in Human Esophageal Adenocarcinoma. Frontiers in Oncology, 12, 903874.

+ Open protocol

+ Expand

Western Blot Analysis of EGFR and α5 Integrin

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in 1% Triton X-100, NaF [100 mmol/L], NaPPi [10 mmol/L], and Na₃VO₄ [1 mmol/L] in PBS, supplemented with complete anti-protease cocktail (Roche, Basel, Switzerland). A total of 10 µg of protein was separated on precast gradient 4–20% SDS-PAGE gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Buckinghamshire, UK). After blocking, membranes were probed with primary antibodies targeting EGFR (D38B1, #4267; Cell Signaling Technology, Danvers, MA, USA), α5 integrin (D7B7G, #98204S; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, Molsheim, France). Immunological complexes were revealed with horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega, Madison, WI, USA) at a 1/10,000 dilution. Revelation was performed with enhanced chemiluminescence (ECL; BioRad) using an LAS4000 imager (GE Healthcare, Dornstadt, Germany). GAPDH was used as housekeeping protein to serve as the loading control for all cell lysate samples. The quantification of non-saturated images was performed with ImageJ software. Analyses were performed on at least three independent experiments.

Cruz Da Silva E., Foppolo S., Lhermitte B., Ingremeau M., Justiniano H., Klein L., Chenard M.P., Vauchelles R., Abdallah B., Lehmann M., Etienne-Selloum N., Dontenwill M, & Choulier L. (2022). Bioimaging Nucleic-Acid Aptamers with Different Specificities in Human Glioblastoma Tissues Highlights Tumoral Heterogeneity. Pharmaceutics, 14(10), 1980.

+ Open protocol

+ Expand

Characterization of MCL-1 and SRSF Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Mia^R cells were grown in 2D, 10 cm dishes and treated with 300 nM UNC-721A dissolved in DMSO for times ranging from 1 to 4 h. The samples were harvested using MIB-MS lysis buffer (above), and determination of protein levels was performed by Bradford analysis as described above. The proteins amounts were normalized and the samples were mixed with 4× sample buffer and 20 μg of each sample applied to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride (PVDF) and the membrane blocked with 5% bovine albumin in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at room temperature. Primary antibodies for MCL-1 (Santa Cruz, Santa Cruz, CA), pSRSF (EMD Millipore, Darmstadt, Germany), tSRSF1 (Thermo Fisher, Waltham, MA), tSRSF6 (Invitrogen, Waltham, MA), PARP (Cell Signaling, Danvers, MA), CDK9 (Santa Cruz), pCDK9 (Cell Signaling), RNA polymerase II (phospho Ser2, Abcam, Cambridge, UK), and β-Actin (Santa Cruz) were diluted in 5% bovine serum albumin (BSA)/TBST, and the membranes were incubated overnight at 4 °C. The membranes were washed three times with TBST, followed by incubation in horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega) in 5% nonfat dry milk in TBST. The blots were imaged using the ChemiDoc Touch Imaging System and ECL reagent (Bio-Rad).

Krulikas L.J., McDonald I.M., Lee B., Okumu D.O., East M.P., Gilbert T.S., Herring L.E., Golitz B.T., Wells C.I., Axtman A.D., Zuercher W.J., Willson T.M., Kireev D., Yeh J.J., Johnson G.L., Baines A.T, & Graves L.M. (2018). Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth. SLAS discovery : advancing life sciences R & D, 23(8), 850-861.

+ Open protocol

+ Expand

Characterization of SPPL3 Monoclonal Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols

Monoclonal antibodies against SPPL3 (clone 7F9), were characterized previously^{20 (link)}. Anti-V5 (mIgG2A, clone R960-25, Life Technologies, Carlsbad, USA), anti-Calnexin (rabbit pAb, Enzo Life Sciences, Farmingdale, USA), anti-TGN46 (sheep, pAb, AP32693PU, Acris, Maryland, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Promega (Madison, USA), and the fluorescent secondary antibodies donkey anti-mouse 488 and donkey anti-sheep 568 were obtained from Thermo Fisher, Waltham, USA.

Papadopoulou A.A., Stelzer W., Silber M., Schlosser C., Spitz C., Haug-Kröper M., Straub T., Müller S.A., Lichtenthaler S.F., Muhle-Goll C., Langosch D, & Fluhrer R. (2022). Helical stability of the GnTV transmembrane domain impacts on SPPL3 dependent cleavage. Scientific Reports, 12, 20987.

+ Open protocol

+ Expand

Quantitative Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell was scraped and centrifuged at 4 °C. Pellets were lysed in RIPA Lysis Buffer System supplemented with 1 × Halt protease inhibitor cocktail and 1 × Halt phosphatase inhibitor cocktail (Santa Cruz Biotechnology). Protein concentrations were measured using Bio‐Rad protein Assay (Bio‐Rad). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to Protran nitrocellulose membranes (Whatman, Boston, MA, USA). Membranes were probed with specific primary antibodies, followed by horseradish peroxidase (HRP)‐conjugated secondary antibodies (Promega). Protein bands were visualized using Amersham ECL western blotting detection reagent (GE Healthcare, Pittsburgh, PA, USA), and images were taken on the ChemiDoc XRS System (Bio‐Rad). The protein bands intensities were semiquantitatively analyzed by densitometry using imagej software (NIH Image).

Hong J., Maacha S, & Belkhiri A. (2018). Transcriptional upregulation of c‐MYC by AXL confers epirubicin resistance in esophageal adenocarcinoma. Molecular Oncology, 12(12), 2191-2208.

+ Open protocol

+ Expand

Western Blot Analysis of DNA Damage Response

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were washed with cold PBS, and then lysed in RIPA buffer (Cell Signaling Technology). After centrifugation at 14,000 rpm for 15 minutes at 4°C, the supernatants were collected, and the protein concentrations were measured using BCA protein assay reagent (BIO-RAD). Subsequently, equal amounts of proteins were separated in NuPAGE 4-12% Bis-Tris gradient gel (Invitrogen #NP0335), and transferred onto nitrocellulose membranes (Invitrogen #B301002). After blocking with 5% milk, the membranes were then probed at 4°C overnight with various primary antibodies: anti-γ-H2AX (Cell Signaling), anti-phospho-Chk1 (Ser345) (Cell Signaling), anti-phospho-Chk2 (Thr68) (Cell Signaling), anti-PRMT5 (Abcam), anti-RPA1 (Abcam), anti-phospho-RPA2 (S4/S8) (Bethyl Laboratories), anti-RPA2 (Ab-2) (Calbiochem), cleaved caspase-3 (Cell Signaling), and anti-β-actin (Sigma), washed with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20; pH 7.6), and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega) at room temperature for 1 hour. Finally, after washing with TBST, the antibody-bound membranes were treated with enhanced chemiluminescent western blot detection reagents (GE Healthcare) and visualized with an x-ray film (GE Healthcare).

Wei X., Yang J., Adair S.J., Kuscu C., Lee K.Y., Kane W.J., O’Hara P.J., Liu D., Demirlenk Y.M., Habieb A., Yilmaz E., Dutta A., Bauer T.W., & Adli M. (2020). Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells.

+ 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?

Revolutionizing how scientists
search and build protocols!