Adherent cells were scraped, and zebrafish tumors were mechanically homogenized in RIPA lysis buffer containing 1:100 protease inhibitors (P8340, Sigma-Aldrich) and 20 µM N-ethylmaleimide (Sigma-Aldrich). Lysates were incubated for 20 min on ice, and spun down for 10 min at 14,000 RPM at 4°C. Samples were denatured by adding Laemmli sample buffer (BioRad) with 5% β-mercaptoethanol (Sigma-Aldrich), and boiled at 95°C for 5 min prior to loading. Protein concentrations were determined using the DC protein assay (BioRad). Proteins (20 µg) were separated on a 4–20% mini-PROTEAN TGX (BioRad) precast gel, and transferred onto a nitrocellulose membrane using the iBlotting system (Invitrogen). Primary antibodies used were: Anti-SATB2 (zebrafish tumors, 1 ug/ml; Ab51502, Abcam), Anti-SATB2 (human cells, 1:200; HPA029543, Sigma Aldrich) and Anti-Beta Actin (1 µg/ml; A2228, Sigma-Aldrich) as a loading control. Additional antibodies used against SATB1 and SATB2 are listed in Figure 2—figure supplement 3B and Figure 3—figure supplement 1A . Protein bands were detected by rabbit anti-mouse HRP (1:20,000, Pierce) or swine anti-rabbit HRP (1:20,000, Pierce).
Hpa029543
Manufactured by Merck Group
HPA029543 is a laboratory equipment product from Merck Group. It is designed to perform specific laboratory functions. No further details on its core functionality or intended use are provided in order to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (2 mentions)
P8340, by Merck Group (2 mentions)
N ethylmaleimide, by Merck Group (2 mentions)
Iblotting system, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx, by Bio-Rad (2 mentions)
Ab51502, by Abcam (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
A2228, by Merck Group (2 mentions)
2 protocols using hpa029543
1
Protein Extraction and Western Blot Analysis
2
SATB2 Expression Analysis in Cells and Zebrafish Tumors
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Adherent cells were scraped, and zebrafish tumors were mechanically homogenized in RIPA lysis buffer containing 1:100 protease inhibitors (P8340, Sigma-Aldrich) and 20 µM N-ethylmaleimide (Sigma-Aldrich). Lysates were incubated for 20 minutes on ice, and spun down for 10 minutes at 14,000 RPM at 4°C. Samples were denatured by adding Laemmli sample buffer (BioRad) with 5% β-mercaptoethanol (Sigma-Aldrich), and boiled at 95°C for 5 minutes prior to loading.
Protein concentrations were determined using the DC protein assay (BioRad). Proteins (20ug)
were separated on a 4-20% mini-PROTEAN TGX (BioRad) precast gel, and transferred onto a nitrocellulose membrane using the iBlotting system (Invitrogen). Primary antibodies used were:
Anti-SATB2 (zebrafish tumors, 1ug/ml; Ab51502, Abcam), Anti-SATB2 (human cells, 1:200; HPA029543, Sigma Aldrich) and Anti-Beta Actin (1ug/ml; A2228, Sigma-Aldrich) as a loading control. Additional antibodies used against SATB1 and SATB2 are listed in Supplementary Figure 5A-B. Protein bands were detected by rabbit anti-mouse HRP (1:20,000, Pierce) or swine antirabbit HRP (1:20,000, Pierce).
Protein concentrations were determined using the DC protein assay (BioRad). Proteins (20ug)
were separated on a 4-20% mini-PROTEAN TGX (BioRad) precast gel, and transferred onto a nitrocellulose membrane using the iBlotting system (Invitrogen). Primary antibodies used were:
Anti-SATB2 (zebrafish tumors, 1ug/ml; Ab51502, Abcam), Anti-SATB2 (human cells, 1:200; HPA029543, Sigma Aldrich) and Anti-Beta Actin (1ug/ml; A2228, Sigma-Aldrich) as a loading control. Additional antibodies used against SATB1 and SATB2 are listed in Supplementary Figure 5A-B. Protein bands were detected by rabbit anti-mouse HRP (1:20,000, Pierce) or swine antirabbit HRP (1:20,000, Pierce).
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