Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total p21 Waf1/Cip1 Sandwich ELISA Kit #7167

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
p21 Waf1/Cip1 Ab Coated Microwells 96 tests
p21 Waf1/Cip1 Detection Ab 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M R

Reactivity Key:  H=Human  M=Mouse  R=Rat
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST's PathScan® Total p21 Waf1/Cip1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total p21 Waf1/Cip1 protein. A p21 Waf1/Cip1 mouse mAb has been coated onto the microwells. After incubation with cell lysates, total p21 Waf1/Cip1 protein is captured by the coated antibody. Following extensive washing, a p21 Waf1/Cip1 antibody is added to detect the captured total p21 Waf1/Cip1 protein. Anti-rabbit IgG, HRP-linked Antibody #7074 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of total p21 Waf1/Cip1 protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total p21 Waf1/Cip1 Sandwich ELISA Kit #7167 detects endogenous levels of total p21 Waf1/Cip1 protein. As shown in Figure 1, cell lysates from 293, HeLa, K562 and THP1 are analyzed using PathScan® Total p21 Waf1/Cip1 Sandwich ELISA Kit #7167. Measured levels of total p21 Waf1/Cip1 protein correlate with levels detected by Western blot analysis. In Figure 3, Western blot analysis of protein captured in the p21 Waf1/Cip1 antibody coated microwell shows a major band corresponding to the p21 Waf1/Cip1 protein.This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

Figure 1: p21 Waf1/Cip1 protein from 293, HeLa, K562 and THP1 cells is detected by PathScan® Total p21 Waf1/Cip1 Sandwich ELISA Kit #7167. The levels of p21 Waf1/Cip1 protein measured using PathScan® Total p21 Waf1/Cip1 Sandwich ELISA Kit #7167 correlate with p21 Waf1/Cip1 protein levels detected by Western blot analysis. Absorbance450 nm is shown in the top figure, while the corresponding Western blot using p21 Waf1/Cip1 Antibody #2946, is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from 293 cells and the absorbance450 nm is shown.

Specificity

Specificity

Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well capture protein. Lysates were prepared from human 293 cells and incubated in wells coated with capture p21 Waf1/Cip1 antibody. Wells were then washed and captured protein was solubilized in SDS gel loading buffer. Western blot analysis of 293 cell starting lysate (lane 1) was performed, using p21 Waf1/Cip1 Antibody #2931. The major band detected in the captured material corresponds to the p21 Waf1/Cip1 protein (lane 2).


Background

The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression. It functions in stoichiometric relationships forming heterotrimeric complexes with cyclins and cyclin-dependent kinases. In association with CDK2 complexes, it serves to inhibit kinase activity and block progression through G1/S (1). However, p21 may also enhance assembly and activity in complexes of CDK4 or CDK6 and cyclin D (2). The carboxy-terminal region of p21 is sufficient to bind and inhibit PCNA, a subunit of DNA polymerase, and may coordinate DNA replication with cell cycle progression (3). Upon UV damage or during cell cycle stages when cdc2/cyclin B or CDK2/cyclin A are active, p53 is phosphorylated and upregulates p21 transcription via a p53-responsive element (4). Protein levels of p21 are downregulated through ubiquitination and proteasomal degradation (5).

  1. Pestell, R.G. et al. (1999) Endocrine Rev. 20, 501-534.
  2. Cheng, J. et al. (1999) EMBO J. 18, 1571-1583.
  3. Flores-Rozas, H. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8655-8659.
  4. Wang, Y. and Prives, C. (1995) Nature 376, 88-91.
  5. Sheaff, R.J. et al. (2000) Cell 5, 403-410.
  6. Pechnick, R.N. et al. (2008) Proc Natl Acad Sci U S A 105, 1358-63.

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