p27 Antibody (F-8): sc-1641

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

The VSMCs were cultured in a 9-cm diameter dish, grown to 70–80% confluency, and then starved in serum-free medium for 24 h. The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase cocktails. Equal amounts of protein (60–100 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked, and then incubated with various antibodies overnight, such as anti-CDK4 (Cat. no. SC23896, 1:500), anti-CDK6 (Cat. no. SC53638, 1:500), anti-p27Kip1 (Cat. no. SC1641, 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-SM22a (Cat. no. A5228, 1:1,000), anti-calponin (Cat. no. C2687, 1:1,000), anti-smooth muscle (SM) α-actin (Cat. no. A2172, 1:1,000) (all from Sigma), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; Tyr183/185; Cat. no. 5135, 1:1,000), anti-JNK (Cat. no. 9258 1:1,000), anti-p38 mitogen-activated protein kinase (MAPK; Cat. no. 9228, 1:1,000), anti-phospho-p38 MAPK (Thr180/Tyr182; Cat. no. 4092, 1:1,000; p-MAPK), anti-extracellular signal-regulated kinase (ERK)1/2 (Cat. no. 4696, 1:1,000), anti-phospho-ERK1/2 (p-ERK, Tyr204; Cat. no. 4374, 1:1000), anti-phospho-Akt (p-Akt, S473; Cat. no. 12694, 1:1,000), anti-Akt (Cat. no. 2920, 1:1,000), anti-phospho-GSK-3β (p-GSK-3β, S9; Cat. no. 14630, 1:1,000), anti-GSK-3β (Cat. no. 9832, 1:1,000) (all from Cell Signaling Technology), anti-ILK (Cat. no. 61183, 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), anti-intercellular adhesion molecule-1 (ICAM-1; Cat. no. 4915, 1:500), anti-vascular cell adhesion molecule-1 (VCAM-1; Cat. no. 13662, 1:500) (both from Cell Signaling Technology), anti-matrix metalloproteinase (MMP)-2 (Cat. no. SC13594, 1:500), anti-MMP-9 (Cat. no. SC21733, 1:500) anti-tissue inhibitors of metalloproteinase (TIMP)-1 (Cat. no. SC21734, 1:500), anti-TIMP-2 (Cat. no. SC365671, 1:500) (all from Santa Cruz Biotechnology, Inc.), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cat. no. 5174, 1:2,500) and anti-β-actin (Cat. no. 8457, 1:2,500) (both from Cell Signaling Technology), and then with the horseradish peroxidase-conjugated secondary antibody (Beijing TDY Biotech Co., Ltd.) (1:5,000) for 2 h. Specific protein expression levels were normalized to GAPDH or β-actin for total protein analyses or to total proteins for phosphorylated protein measurements. The blots were analyzed using the ChemiDoc™ MP imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiments were replicated a number of times.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Western blotting CDKN1B using p27 Antibody (F-8): sc-1641 from Santa Cruz Biotechnology.

Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for p27 Antibody (F-8): sc-1641 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Western blotting CDKN1B using p27 Antibody (F-8): sc-1641 from Santa Cruz Biotechnology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.