Hrp conjugated β actin antibody

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The HRP-conjugated β-actin antibody is a lab equipment product that provides a detection solution for the β-actin protein. This antibody is conjugated with horseradish peroxidase (HRP), which enables direct visualization and quantification of the target protein in various applications.

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Lab products found in correlation

Close spelling variants of this product

β-actin (5071 citations) β-actin antibody (369 citations) HRP-conjugated β-actin (25 citations) β-actin-HRP (23 citations) Actin-HRP (17 citations) HRP-conjugated actin (4 citations) Actin- HRP antibody (4 citations) HRP-conjugated anti-β-actin antibody (4 citations) HRP-conjugated mouse anti-β-actin antibody (2 citations)

7 protocols using hrp conjugated β actin antibody

Western Blot Antibody Reagents

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Rabbit anti-cytosolic PEPCK was prepared in our laboratory [18 (link)]. Goat anti-muscle pyruvate kinase (PK-M) was from Rockland Immunochemicals Inc. (Gilbertsville, PA). An HRP-conjugated β-actin antibody was from Santa Cruz Biotechnology (sc-47778-HRP; Santa Cruz, CA). Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) was from Cell Signaling Technology Inc. (#4370, Beverly, MA). Secondary antibodies were goat anti-rabbit IgG-HRP, rabbit anti-goat IgG-HRP (Jackson ImmunoResearch, West Grove, PA), and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR).

Bertinat R., Westermeier F., Silva P., Gatica R., Oliveira J.M., Nualart F., Gomis R, & Yáñez A.J. (2018). The Antidiabetic Agent Sodium Tungstate Induces Abnormal Glycogen Accumulation in Renal Proximal Tubules from Diabetic IRS2-Knockout Mice. Journal of Diabetes Research, 2018, 5697970.

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Protein Isolation and Western Blot Analysis

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Protein lysates from treated cells or aortic tissues were prepared as previously described^{19 (link)}. Nuclear and cytoplasmic proteins were isolated using NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Protein samples (15 µg per lane) were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and were transferred to PVDF membranes. The membranes were blocked for 1 h in blocking solution comprising Tris-buffered saline containing 5% nonfat dried milk and 0.5% Tween 20 and then were incubated with a primary antibody against ADAMTS-4 PARP-1, cleaved PARP (Asp214) (9548, Cell Signaling), cleaved caspase-3 (9661, Cell Signaling), or versican degradation product. The blots were then washed with PBS with tween, incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Cell Signaling), and developed with Clarity Enhanced Chemiluminescence (ECL; Bio-Rad). The blots were exposed with HyBlot ES autoradiography film (Denville Scientific Inc., Holliston, MA) and quantified by using Image J Software (National Institutes of Health, Bethesda, MD). We confirmed equal protein loading by immunoblotting with HRP-conjugated β-actin antibody (Santa Cruz) or GAPDH antibody (Santa Cruz), β-tubulin, or lamin B1 (Cell Signaling).

Ren P., Hughes M., Krishnamoorthy S., Zou S., Zhang L., Wu D., Zhang C., Curci J.A., Coselli J.S., Milewicz D.M., LeMaire S.A, & Shen Y.H. (2017). Critical Role of ADAMTS-4 in the Development of Sporadic Aortic Aneurysm and Dissection in Mice. Scientific Reports, 7, 12351.

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Western Blot Analysis of Liver GAMT

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General preparation of the protein samples and western blotting were carried out as described.⁴³ (link) Briefly, liver specimens were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing Halt Protease Inhibitor Cocktail (cat. no. 78430, ThermoFisher, Waltham, MA) to isolate proteins. Total protein extract, 50 μg, quantified with Bio-Rad Protein Assay Dye (cat. no. 5000006, BioRad, Hercules, CA), were separated by SDS-PAGE and probed with human GAMT antibody (Abcam, Cambridge, UK, cat. no. ab126736; 1:1,000 dilution). HRP-conjugated β-actin antibody (Santa Cruz Biotechnology, Dallas, TX, cat. no. sc-47778; 1:5,000) was used as loading control. hGAMT was labeled by HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, cat. no. sc-2004; 1:5,000), and targeted proteins were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (cat. no. PI34579, ThermoFisher).

Khoja S., Lambert J., Nitzahn M., Eliav A., Zhang Y., Tamboline M., Le C.T., Nasser E., Li Y., Patel P., Zhuravka I., Lueptow L.M., Tkachyova I., Xu S., Nissim I., Schulze A, & Lipshutz G.S. (2022). Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities. Molecular Therapy. Methods & Clinical Development, 25, 278-296.

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Immunoblotting of CRC Cell Signaling

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CRC cells were treated with CM and/or seribantumab for 30 minutes. Cell lysates were processed and run through SDS-PAGE gel electrophoresis as described previously (7 (link),18 (link)). An HRP-conjugated β-actin antibody was obtained from Santa Cruz Biotechnology and was used in 1:8000 dilution ((Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189, Santa Cruz, CA, USA). All other antibodies were from Cell Signaling Technology and were used in 1:1000 dilutions (Beverly, MA, USA). Phospho- AKT-S473 (Cell Signaling Technology Cat# 9271, RRID:AB_329825), total AKT (Cell Signaling Technology Cat# 9272, RRID:AB_329827), Phospho-HER3-Y1289 (Cell Signaling Technology Cat# 2842, RRID:AB_11178795), total HER3 (Cell Signaling Technology Cat# 12708, RRID:AB_2721919). For each experiment, protein lysates were loaded into multiple gels with equal quantities and processed at the same time for separately probing for antibodies specific to phosphorylated proteins and total proteins. All membranes were probed with β-actin as loading controls and a representative loading control was used in the figures. Western blotting images were developed by Enhanced chemiluminescent (ECL) substrate and autoradiography films ( both from Thermo Fisher Scientific), and figures presented representative results from one experiment of at least three independent experiments.

Rathore M., Zhang W., Wright M., Bhattacharya R., Fan F., Vaziri-Gohar A., Winter J., Wang Z., Markowitz S.D., Willis J., Ellis L.M, & Wang R. (2022). Liver Endothelium Promotes HER3-mediated Cell Survival in Colorectal Cancer with Wild-type and Mutant KRAS. Molecular cancer research : MCR, 20(6), 996-1008.

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Stem Cell Factor Signaling Analysis

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Recombinant human stem cell factor (SCF) was purchased from ORF genetics (Kópavogur, Iceland). Anti-KIT antibody KitC1 was purified as described before [32 (link)]. PE conjugated anti-KIT antibody was purchased from Biolegend (San Diego, CA). PI3 kinase subunit p85α antibody, phospho-Erk Thr202/Tyr204 antibody, Akt antibody, Erk antibody and HRP conjugated β-actin antibody were purchased from Santa Cruz Biotechnology (Dallas, TX). Phospho-Akt (Ser473) antibody was purchased from Cell Signaling Technology (Danvers, MA). Anti-pY antibody 4G10 and chemiluminescent HRP substrate were purchased from Millipore (Billerica, MA). HRP conjugated goat anti-mouse IgG antibody, HRP conjugated goat anti-rabbit antibody and HRP conjugated donkey anti-goat IgG were purchased from Bioss Antibodies (Beijing, China). KIT inhibitor Imatinib and PI3 kinase inhibitor Copanlisib were purchased from MedChemExpress (Monmouth Junction, NJ).

Zhu G., Shi J., Zhang S., Guo Y., Huang L., Zhao H., Jiang Y, & Sun J. (2020). Loss of PI3 kinase association improves the sensitivity of secondary mutation of KIT to Imatinib. Cell & Bioscience, 10, 16.

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Western Blot Antibody Preparation

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Phosphor-tyrosine antibody 4G10 was purchased from Millipore (Billerica, MA), KIT antibody was described previously [18 (link)], HRP conjugated β-actin antibody, HRP conjugated goat anti-mouse IgG antibody, and HRP conjugated goat anti-rabbit antibody were from Santa Cruz Biotechnology (Dallas, TX), FLAG antibody was from Sigma (St. Louis, MO). Lipofectamine 2000 and protein G-sepharose beads were from Thermofisher scientific (Waltham, MA), chemiluminescent HRP substrate was from Millipore (Billerica, MA). Human KIT cDNA was described previously [19 (link)], human PTPRE cDNA in pcDNA3.1 with FLAG tag at 3′ end was ordered from FenghuiBio (Changsha, China). QuikChange mutagenesis kit was purchased from Agilent (La Jolla, CA) and used according to the manufacturer's instructions. Recombinant human stem cell factor (SCF) was from ORF genetics (Kópavogur, Iceland).

Zhang S., Zhang L., Jiang Z., Guo Y., Zhao H, & Sun J. (2021). Protein tyrosine phosphatase receptor type E (PTPRE) regulates the activation of wild-type KIT and KIT mutants differently. Biochemistry and Biophysics Reports, 26, 100974.

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Stripping and Reprobing Blot

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Restore Western Blot Stripping buffer (Thermo Scientific) for 15 min at room temperature, blocked for 1 h, and then reprobed with an HRP conjugated β-actin antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA).

Jiang R, & Lönnerdal B. (2017). Bovine lactoferrin and lactoferricin exert antitumor activities on human colorectal cancer cells (HT-29) by activating various signaling pathways. Biochemistry and cell biology = Biochimie et biologie cellulaire, 95(1).

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