Frozen tumor samples were diced in ice-cold lysis buffer (1% NP-40, 50 mM Tris-HCl pH 8.0, 100 mM sodium fluoride, 30 mM sodium pyrophosphate, 2 mM sodium molybdate, 5 mM EDTA, 2 mM sodium orthovanadate) on dry ice and homogenized with a Tissue Tearor Homogenizer for 3 seconds, 3–5 times, on ice, and the cell lysate was then rocked overnight at 4°C. Lysates were cleared by centrifugation at 14,000 rpm for 30 min at 4°C, and lysate protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad Laboratories Hercules, CA, USA). Electrophoresis and western blotting were performed using standard techniques. The hybridization signals were detected by chemiluminescence (Immobilon Western, Millipore Corporation, MA) and captured using a FUJI LAS1000-plus chemiluminescence imaging system (Fuji Film, Tokyo, Japan). Primary antibodies were DYS1 (Novocastra, NCL-DYS1, raised against the dystrophin rod domain, amino acids 1181 and 1388, detects 427 kDa dystrophin isoform), DYS2 (Novocastra, NCL-DYS2, raised against the C-terminal 17 amino acids of dystrophin, detects 240 kDa mini-dystrophin), 7A10 (Santa Cruz, sc-47760, raised against amino acids 3200–3684 of dystrophin, detects Dp71), and GAPDH (Sigma, G8795).
Sc 47760
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-47760 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for scientific research applications. The core function of Sc-47760 is to [CORE FUNCTION DESCRIPTION].
Automatically generated - may contain errors
Lab products found in correlation
Ncl dys1, by Leica Biosystems (2 mentions)
Immobilon western, by Merck Group (2 mentions)
Las1000 plus chemiluminescence imaging system, by Fujifilm (2 mentions)
Ncl dys2, by Leica Biosystems (2 mentions)
Gapdh, by Merck Group (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Sample buffer, by Bio-Rad (1 mentions)
Ab14748, by Abcam (1 mentions)
Sc 2027, by Santa Cruz Biotechnology (1 mentions)
3 protocols using sc 47760
1
Dystrophin Isoform Detection in Frozen Tissues
2
Dystrophin Isoform Detection in Frozen Tissues
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Frozen tumor samples were diced in ice-cold lysis buffer (1% NP-40, 50 mM Tris-HCl pH 8.0, 100 mM sodium fluoride, 30 mM sodium pyrophosphate, 2 mM sodium molybdate, 5 mM EDTA, 2 mM sodium orthovanadate) on dry ice and homogenized with a Tissue Tearor Homogenizer for 3 seconds, 3–5 times, on ice, and the cell lysate was then rocked overnight at 4°C. Lysates were cleared by centrifugation at 14,000 rpm for 30 min at 4°C, and lysate protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad Laboratories Hercules, CA, USA). Electrophoresis and western blotting were performed using standard techniques. The hybridization signals were detected by chemiluminescence (Immobilon Western, Millipore Corporation, MA) and captured using a FUJI LAS1000-plus chemiluminescence imaging system (Fuji Film, Tokyo, Japan). Primary antibodies were DYS1 (Novocastra, NCL-DYS1, raised against the dystrophin rod domain, amino acids 1181 and 1388, detects 427 kDa dystrophin isoform), DYS2 (Novocastra, NCL-DYS2, raised against the C-terminal 17 amino acids of dystrophin, detects 240 kDa mini-dystrophin), 7A10 (Santa Cruz, sc-47760, raised against amino acids 3200–3684 of dystrophin, detects Dp71), and GAPDH (Sigma, G8795).
3
Dynabeads Co-Immunoprecipitation of Dystrophin
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Immunoprecipitation was investigated using the Dynabeads® Co-Immunoprecipitation (Co-IP) kit (Novex, Thermofisher, Scientific Inc., Boston, MA, USA). The mouse anti-ATP5A (2 μg; ab14748; Abcam, Cambridge, UK) or mouse anti-dystrophin (7A10) antibody (20 μg; sc-47760, Santa Cruz Biotechnology Inc., CA, USA) that detects an epitope corresponding to amino acids 3558–3684 of dystrophin was coupled with 10 mg of Dynabeads® M-270 Epoxy overnight at 4 °C. The antibody coupled beads were washed and extracted in a magnetic field. 1.5 mg of antibody coupled beads was mixed with 1g of total protein lysate derived from DMD3-derived primary cultured myocytes for dynabeads Co-IP complex preparation as specified by the protocol. The protein lysate for this assay was prepared with lysis buffer containing 0.1% of SDS. The complexes were washed, and the purified protein was separated through a magnetic field and transferred to a clean tube. A fraction was mixed with sample buffer (BioRad Laboratories Inc., Hercules, CA, USA), boiled for 5 min at 95 °C and used for SDS-PAGE and western blot to probe for dystrophin and MT proteins. To investigate for non-specific binding, a normal rabbit IgG (sc-2027; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was also used as a western blotting control.
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