Human Notch-1 Intracellular Domain Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all the solutions and reagents to room temperature before use; otherwise the detection limit may be compromised.	-Sheep (1:200) -Always wear gloves or use forceps when handling blotting membranes to avoid contamination with protein from fingers.

Upstream tips
-Bring all the solutions and reagents to room temperature before use; otherwise the detection limit may be compromised.
Protocol tips
-Sheep (1:200) -Always wear gloves or use forceps when handling blotting membranes to avoid contamination with protein from fingers.

Publication protocol

Cells were lysed in RIPA-B buffer (20 mM Na2HPO4 pH 7.4, 150 mM NaCl and 1% Triton X-100) supplemented with Protease Inhibitor Cocktail (Roche) for 30 min on ice with occasional vortexing, followed by 30 min centrifugation at 13,000 relative centrifugal force at 4 °C. For CDKpY15 detection, lysis buffer was supplemented with 1 mM dithiothreitol, 50 mM NaF, 30 mM tetrasodium pyrophosphate, 0.1 mM sodium orthovanadate, 10 mM β-glycerophosphate and 15 mM para-nitrophenylphosphate. The BCA Protein Assay Kit (Thermo Scientific, catalogue number 23225) was used to determine protein concentration. Next, 20–75 μg protein were separated by SDS–PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with TBST (10 mM Tris-HCl pH 7.9, 150 mM NaCl and 0.05% Tween-20) containing 3% skim milk, incubated with primary antibodies overnight, washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham), washed three times with TBST and subsequently reacted with ECL or ECL Prime (GE Healthcare). Luminescence was detected with X-ray films, which were scanned, or using the Bio-Rad ChemiDoc MP System. Blots were processed digitally by adjusting the brightness and contrast, and by rotating and cropping, when necessary. The following primary antibodies were used: rabbit anti-GFP (Invitrogen, catalogue number A-6455, 1:500), mouse anti-CHK1 (FL-393, Santa Cruz Biotechnology, catalogue number sc-8408, 1:1,000), mouse anti-β-actin (Sigma, catalogue number A2066, 1:4,000), rabbit anti-p53 (DO-1, Santa Cruz Biotechnology, catalogue number sc-6243, 1:1,000), mouse anti-p53 (Santa Cruz Biotechnology, catalogue number sc-126, 1:500), rabbit anti-p21 (Santa Cruz Biotechnology, catalogue number sc-397, 1:500), rabbit anti-NANOG (Millipore, catalogue number AB5731, 1:1,000), mouse anti-HA (Abcam, catalogue number ab16918, 1:4,000), rabbit anti-phospho Cdc2 (CDK1) Tyr15 (Cell Signaling, catalogue number 9111, 1:500), mouse anti-CDC25A (Santa Cruz Biotechnology, catalogue number sc-7389, 1:250), sheep anti-Notch-1 Intracellular Domain (R&D Systems, catalogue number AF3647, 1:200) and mouse anti-α-Tubulin (Sigma, catalogue number T9026, 1:1,500). Quantification of protein level was performed using the ImageJ software.

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Manufacturer protocol

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