Cells were collected by trypsinization, then washed with PBS (Biological Industries, Israel). Proteins were extracted with 100 μL in-house made RIPA (1% NP-40, 0.1%SDS, 1 M Tris-HCL pH 7.4, NaCl, Sodium deoxycholate, EDTA, and water) + Protease Inhibitors (Roche, USA) and quantified using Bradford reagents (Sigma Aldrich, USA). Extracted proteins were reconstituted in an in-house Protein Sample Buffer (Glycerol 100%, 1 M Tris pH6.8, 10% SDS, β- mercaptoethanol, bromophenol blue) and heated for 10 min at 95 °C. Proteins were separated on 10% SDS-PAGE gels and transferred to 0.22 µm PVDF membrane (FroggaBio, USA) by an electric current at 300 mA for 70 min. The membrane was blocked in 5% BSA/TBST for 2 h, then incubated with primary antibody overnight. Scd1 antibody was purchased from Cell Signaling Technology USA, (#2794, 1:1,000). The membrane was washed, then incubated with HRP conjugated secondary antibody (Abcam, USA, Ab 6721, 1:10,000). eIF4G2/p97 antibody from Cell Signaling Technology (#2182, 1: 3000), or mouse monoclonal anti-α-tubulin antibody (T5168; Sigma Aldrich, USA) were used as a loading control. Chemiluminescence was detected by ECL (Advansta, USA), using Molecular Imager ChemiDocTM XRS (Bio-Rad, USA).
Scd1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The SCD1 antibody is a primary antibody that recognizes the Stearoyl-CoA Desaturase 1 (SCD1) protein. SCD1 is an enzyme that catalyzes the rate-limiting step in the synthesis of monounsaturated fatty acids. This antibody can be used to detect and study the SCD1 protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Sartorius (1 mentions)
Mouse monoclonal anti α tubulin antibody, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
β actin, by Proteintech (1 mentions)
Fbw7 antibody, by Fortis Life Sciences (1 mentions)
Nr4a1 antibody, by Abcam (1 mentions)
Quantity one program, by Bio-Rad (1 mentions)
3 protocols using scd1 antibody
1
Western Blot Analysis of Protein Expression
2
Comprehensive Western Blot Analysis
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Western blotting was carried out as previously described [5 (link)]. Briefly, whole-cell protein lysates were extracted and then were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Bio-Rad). After blocking, the membranes were incubated with corresponding antibodies: FBW7 antibody (Bethyl), SCD1 antibody (Cell Signaling Technology), NR4A1 antibody (Abcam), NR4A2 antibody (Abcam), β-actin (Proteintech). Next, the membranes were probed with secondary antibodies conjugated to HRP (Proteintech). Finally, immunoblots were incubated with an enhanced chemiluminescence detection kit (Millipore) and visualized by imaging systems (CLiNX).
3
SCD1 Protein Expression Quantification
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For the detection of SCD1, the total protein from the 3T3-L1 cells was extracted using a protein extraction buffer (50 mM NaF, 50 mM Tris HCl, 5 mM NaPPi, 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, and 1 mM EDTA). For each sample, 40 μg of protein was denatured in Laemmli Buffer for 5 minutes at 95°C. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with a blocking buffer (5% skim milk in PBS), the membranes were incubated with the SCD1 antibody (Cell Signaling Technology, Inc., Danvers, MA, USA). The proteins were visualized using enhanced chemiluminescence with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G. Blots were scanned and analyzed using a multiple image analyzer and the Quantity One program (Bio-Rad Laboratories).
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