Secondary antibodies conjugated to peroxidase

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Secondary antibodies conjugated to peroxidase are lab equipment used to detect and amplify signal in various immunoassays. They bind to primary antibodies and catalyze a colorimetric or chemiluminescent reaction, allowing for visualization and quantification of target analytes.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (6 mentions) P2714, by Merck Group (2 mentions) Trans blot sd, by Bio-Rad (1 mentions) Pd l1, by Proteintech (1 mentions) Pierce ecl plus, by Thermo Fisher Scientific (1 mentions) Anti gapdh, by Santa Cruz Biotechnology (1 mentions) Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Thermo Fisher Scientific (1 mentions) Immun blot low fluorescence pvdf membrane, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Sds page, by Thermo Fisher Scientific (1 mentions) Anti flag, by Merck Group (1 mentions) Ecl plus, by GE Healthcare (1 mentions) Anti β actin, by Merck Group (1 mentions)

4 protocols using secondary antibodies conjugated to peroxidase

Western Blot Analysis of Signaling Proteins

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For western-blot analysis, 50 μg of protein from in vivo tissue lysates were resolved by gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes by semi-dry transfer (Biorad Trans-blot SD, Hercules, CA, USA). Blots were blocked with 4% dry powder milk or BSA for 1 h and then incubated with primary antibodies for p-EGFR (Y845), p-EGFR (Y1068), p-EGFR (Y1173), total EGFR, p-Src (Y418), total Src, pSTAT3 (Y705), total STAT3, pSTAT5 (Y694/Y699), total STAT5, COX-2, Cyclin D1, and Cyclin D2, IFN-γ, TLR-4, phospho-p38, IFN-β. These antibodies were all acquired from Santa Cruz Biotech (Santa Cruz, CA, USA); PD-L1 was acquired from Proteintech (Rosemont, IL). Blots were incubated with primary antibodies at 4°C overnight and after washing were incubated for 1 h at room temperature with respective secondary antibodies conjugated to peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). Blots were then developed with an ECL detection system. Densitometric analysis was performed using ImageJ 1.x software [42 (link)].

Mudd A.M., Gu T., Munagala R., Jeyabalan J., Fraig M., Egilmez N.K, & Gupta R.C. (2021). Berry anthocyanidins inhibit intestinal polyps and colon tumors by modulation of Src, EGFR and the colon inflammatory environment. Oncoscience, 8, 120-133.

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Quantification of UGDH Protein Levels

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Cells were lysed using ice-cold RIPA buffer (250 mM Tris, pH: 7.5; 150 mM NaCl; 1% NP-40; 0.5% Na deoxycholate; protease inhibitors P2714 [Sigma-Aldrich, USA]). The total protein concentration of cell lysates was determined using the BCA Protein assay Kit (Thermo Fisher Scientific, USA). Sixty micrograms of total proteins were reduced in Laemeli loading buffer, denatured at 95 °C for 10 min, separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen, Germany) electrophoresis and transferred onto Immun-Blot® Low Fluorescence PVDF Membranes (BIORAD). Protein detection was performed using anti-UGDH (1:500, Sigma-Aldrich, USA, HPA036657) and anti-GAPDH (1:2000, Santa Cruz Biotechnology Inc., USA, SC 47724) antibodies. Secondary antibodies conjugated to peroxidase (1:4000, Santa Cruz Biotechnology Inc., USA) were used and blots were developed using an enhanced chemiluminescence system, Pierce™ ECL Plus (Thermo Scientific), followed by detection on autoradiographic films.

Hengel H., Bosso-Lefèvre C., Grady G., Szenker-Ravi E., Li H., Pierce S., Lebigot É., Tan T.T., Eio M.Y., Narayanan G., Utami K.H., Yau M., Handal N., Deigendesch W., Keimer R., Marzouqa H.M., Gunay-Aygun M., Muriello M.J., Verhelst H., Weckhuysen S., Mahida S., Naidu S., Thomas T.G., Lim J.Y., Tan E.S., Haye D., Willemsen M.A., Oegema R., Mitchell W.G., Pierson T.M., Andrews M.V., Willing M.C., Rodan L.H., Barakat T.S., van Slegtenhorst M., Gavrilova R.H., Martinelli D., Gilboa T., Tamim A.M., Hashem M.O., AlSayed M.D., Abdulrahim M.M., Al-Owain M., Awaji A., Mahmoud A.A., Faqeih E.A., Asmari A.A., Algain S.M., Jad L.A., Aldhalaan H.M., Helbig I., Koolen D.A., Riess A., Kraegeloh-Mann I., Bauer P., Gulsuner S., Stamberger H., Ng A.Y., Tang S., Tohari S., Keren B., Schultz-Rogers L.E., Klee E.W., Barresi S., Tartaglia M., Mor-Shaked H., Maddirevula S., Begtrup A., Telegrafi A., Pfundt R., Schüle R., Ciruna B., Bonnard C., Pouladi M.A., Stewart J.C., Claridge-Chang A., Lefeber D.J., Alkuraya F.S., Mathuru A.S., Venkatesh B., Barycki J.J., Simpson M.A., Jamuar S.S., Schöls L, & Reversade B. (2020). Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy. Nature Communications, 11, 595.

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Western Blot Analysis of YRDC Protein

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Primary dermal fibroblasts were solubilized using ice-cold RIPA buffer (10 mM Tris, pH: 8.0; 150 mM NaCl; 1 mM EDTA; 1% NP-40; protease inhibitors P 2714 [Sigma-Aldrich, USA]). The total protein concentration of extracts was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). 15–25 µg of total cell lysates were separated by Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad, Germany) and blotted onto nitrocellulose membranes (Bio-Rad, Germany). Protein detection was performed using an antibody to YRDC (Santa Cruz Biotechnology Inc., USA). Secondary antibodies conjugated to peroxidase (Santa Cruz Biotechnology Inc., USA) were used and blots were developed using an enhanced chemiluminescence system, Clarity Max Western (Bio-Rad, Germany), followed by detection using the ChemiDoc™ Touch Imaging System (Bio-Rad, Germany).

Schmidt J., Goergens J., Pochechueva T., Kotter A., Schwenzer N., Sitte M., Werner G., Altmüller J., Thiele H., Nürnberg P., Isensee J., Li Y., Müller C., Leube B., Reinhardt H.C., Hucho T., Salinas G., Helm M., Jachimowicz R.D., Wieczorek D., Kohl T., Lehnart S.E., Yigit G, & Wollnik B. (2021). Biallelic variants in YRDC cause a developmental disorder with progeroid features. Human Genetics, 140(12), 1679-1693.

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Western Blot Analysis of γH2AX

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Cells were solubilized by using ice-cold RIPA buffer [10 m Tris, pH: 8.0; 150 m NaCl; 1 m EDTA; 10 m NaF; 1 m Na 3 VO 4 ; 10 µM Na 2 MoO 4 ; 1% NP-40; protease inhibitors P 2714 (Sigma-Aldrich, USA)]. The total protein concentration of extracts was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). 13-25 µg of total cell lysates were separated by 4-12% SDS-PAGE (Invitrogen, Germany) and blotted onto nitrocellulose membranes (GE Healthcare, Germany). Protein detection was performed using phospho-specific antibodies to γH2AX (Ser139) (Cell Signaling Technology, USA). Antibodies to H2AX were purchased from Calbiochem (USA). Anti-β-Actin, anti-FLAG and anti-XRCC4 antibodies were purchased from Sigma-Aldrich. Secondary antibodies conjugated to peroxidase (Santa Cruz Biotechnology Inc., USA) were used and blots were developed using an enhanced chemiluminescence system, ECL Plus (GE Healthcare), followed by detection on autoradiographic films.

Rosin N., Elcioglu N.H., Beleggia F., Isgüven P., Altmüller J., Thiele H., Steindl K., Joset P., Rauch A., Nürnberg P., Wollnik B, & Yigit G. (2015). Mutations in XRCC4 cause primary microcephaly, short stature and increased genomic instability. Human molecular genetics, 24(13).

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