Sc 12462 r

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

Sc-12462-R is a lab equipment product from Santa Cruz Biotechnology. It is designed for use in scientific research applications. The core function of this product is to [DESCRIPTION_NOT_AVAILABLE].

Automatically generated - may contain errors

Lab products found in correlation

Ab7046, by Abcam (2 mentions) Sc 58414, by Santa Cruz Biotechnology (2 mentions) Nb100 64754, by Novus Biologicals (2 mentions) Rsv glycoprotein g, by Bio-Rad (2 mentions) Pbs tween, by Thermo Fisher Scientific (2 mentions) Odyssey clx imaging system, by LI COR (2 mentions) Wbkls0500, by Merck Group (2 mentions) Ab34710, by Abcam (1 mentions) Ab157210, by Abcam (1 mentions) Ab7046, by Santa Cruz Biotechnology (1 mentions) Sc 47778, by Santa Cruz Biotechnology (1 mentions) Sc 1647, by Cell Signaling Technology (1 mentions) P erk, by Cell Signaling Technology (1 mentions)

5 protocols using sc 12462 r

Exosomal Biomarkers in Lung Transplant

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Pooled exosomes isolated from stable LTxR and from those with BOS were analyzed for the presence of SAg (ie, Col-V, Kα1T, Col-IV and Fibronectin) and MHC-II molecules. Ab specific to MHC-II (Abcam ab157210), Col-I (Abcam ab34710), Col-V (Abcam ab7046), and Kα1T (sc-12462-R, Santa Cruz Biotechnology), were used per manufacturer’s suggestion.

Gunasekaran M., Sharma M., Hachem R., Bremner R., Smith M.A, & Mohanakumar T. (2018). Circulating exosomes with distinct properties during chronic lung allograft rejection. Journal of immunology (Baltimore, Md. : 1950), 200(8), 2535-2541.

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ELISA Quantification of Lung Antibodies

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Sera collected on days 10, 20, and 30 were used to measure Ab to the lung SAg, Col-V and Kα1T, by ELISA developed in our laboratory and detailed in earlier publications (8 (link)). Briefly, 1μg/ml Col-V (Sigma C3657) and Kα1T (prepared in the laboratory) were dissolved in 1X PBS and were coated on 96-well ELISA plates (Corning #3369) and stored at 4°C overnight. Sera were diluted (1:100) and were added to the wells and incubated at room temperature for 2 hours. Plates were then washed with PBS-tween (PBST 20), and 100 μl of goat-anti-mouse conjugated HRP (1:10000) was added to the wells. Thereafter, plates were developed with substrate tetra methyl benzidinec (Millipore ES022), and enzymatic reaction was stopped with 1N HCL. The colorimetric reading was obtained with ELISA reader at a 450nm wavelength. Finally, the Ab concentration of the sera was calculated with known concentrations of Ab to Col-V (Abcam ab7046) and Ka1T (Santa Cruz Biotechnology sc-12462-R) performed as standards in the same plates. Sera collected from naive C57BL/6 mice were used as negative control.

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Protein Extraction and Western Blot Analysis

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Total cellular protein was extracted and normalised as previously described (Khodarev et al, 2004 (link)). Protein concentrations were adjusted to 1 mg ml⁻¹ and equal amounts of protein were loaded in each well. For total CXCL10, CXCR3 and ERK proteins, 20–25 μg of proteins per well were loaded, whereas for phosphorylated proteins (pERK), 30–35 μg of proteins per well were loaded. Proteins were separated on 7.5%–10% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. For loading control, we used antibodies for β-actin and α-tubulin (sc-47778 and sc-12462-R; Santa Cruz, Santa Cruz, CA, USA). The CXCL10 and CXCR3 antibodies were purchased from R&D Systems (AF-466-NA and MAB160–100), ERK from Santa Cruz (sc-1647) and pERK from Cell Signaling (Danvers, MA, USA). Images were quantified using ImageJ by integration of pixel values across the area of specific bands.

Wightman S.C., Uppal A., Pitroda S.P., Ganai S., Burnette B., Stack M., Oshima G., Khan S., Huang X., Posner M.C., Weichselbaum R.R, & Khodarev N.N. (2015). Oncogenic CXCL10 signalling drives metastasis development and poor clinical outcome. British Journal of Cancer, 113(2), 327-335.

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Exosomal Viral Antigen Detection

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Immunoblot was used to detect SAgs, 20S proteasome, and viral antigens in exosomes from LTxRs diagnosed with RVI and from stable controls. Total exosome protein (3 µg) was resolved in polyacrylamide gel electrophoresis, and the proteins were transferred into a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk in 1x phosphate buffered saline and was probed with exosome-specific marker CD9 (312102, BioLegend), Col-V (ab7046, Abcam, Cambridge, United Kingdom), and Kα1T (sc-12462-R, Santa Cruz Biotechnology, Dallas, TX). 20S proteasome subunit α3 (sc-58414, Santa Cruz Biotechnology), rhinovirus VP3 (MA5-18249, Thermo Fisher Scientific, Waltham, MA), coronavirus (NB100-64754, Novus Biologicals, Littleton, CO), and RSV glycoprotein G (7950-0980, Bio-Rad Laboratories, Hercules, CA) were used as primary Abs; secondary Abs conjugated with horseradish peroxidase (HRP) were used specific to primary Ab. The blots were washed with PBS Tween (Thermo Fisher Scientific), developed using chemiluminescent HRP substrate (WBKLS0500, MilliporeSigma, Burlington, MA), and exposed using Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE). The band intensity of target protein was quantified using ImageJ software and normalized with CD9.

Gunasekaran M., Bansal S., Ravichandran R., Sharma M., Perincheri S., Rodriguez F., Hachem R., Fisher C.E., Limaye A.P., Omar A., Smith M.A., Bremner R.M, & Mohanakumar T. (2020). Respiratory viral infection in lung transplantation induces exosomes that trigger chronic rejection. The Journal of Heart and Lung Transplantation, 39(4), 379-388.

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Exosomal Biomarkers for Respiratory Viral Infections

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Immunoblot was used to detect SAgs, 20S proteasome, and viral antigens in exosomes from LTxRs diagnosed with RVI and from stable controls. Total exosome protein (3μg) was resolved in polyacrylamide gel electrophoresis, and the proteins were transferred into a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in 1X PBS and was probed with exosome-specific marker CD9 (312102, Biolegend, SanDiego, CA), Col-V (ab7046, Abcam), and Kα1T (sc-12462-R, SantaCruz Biotechnology, Dallas, TX). 20S proteasome subunit α3 (sc-58414, SantaCruz Biotechnology), rhinovirus VP3 (MA5–18249, Thermo Fisher Scientific, Waltham, MA), coronavirus (NB100–64754, Novus Biologicals, Littleton, CO), RSV Glycoprotein G (7950–0980, Bio-Rad Laboratories, Hercules, CA) were used as primary Abs; secondary Abs conjugated with HRP were used specific to primary Ab. The blots were washed with PBS Tween (Thermo Fisher Scientific), developed using chemiluminescent HRP substrate (WBKLS0500, MilliporeSigma), and exposed using Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE). The band intensity of target protein was quantified using ImageJ software and normalized with CD9.

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