Figure 1: Phospho-SLP-76 (Ser376) protein from untreated (-) and H2O2 treated (+) Jurkat cells is detected by PathScan® Phospho-SLP-76 (Ser376) Sandwich ELISA kit #78222. A lower signal of phospho-SLP-76 protein is seen in the untreated lysate. However, similar levels of total SLP-76 protein from both untreated and treated lysates are shown by Western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using SLP-76 Rabbit Antibody #4958 (left panel) or Phospho-SLP-76 (Ser376) (D9D6E) Rabbit mAb #14745 (right panel), are shown in the bottom figure.Learn more about how we get our images
Figure 2: The relationship between protein concentration of lysates from untreated and H2O2-treated Jurkat cells and the absorbance at 450 nm as detected by the PathScan® Phospho-SLP-76 Ser376 Sandwich ELISA Kit is shown. Unstarved Jurkat cells (0.5-1.0 x 106 cells/ml) were treated for 3 min with H2O2 (11 mM at 37°C), or left unteated, and then lysed with Cell Lysis Buffer (#9803).Learn more about how we get our images
|Product Includes||Volume||Solution Color|
|SLP-76 Rabbit mAb Coated Microwells||96 tests|
|Phospho-SLP-76 (Ser376) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X)||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
The PathScan® Phospho-SLP-76 (Ser376) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of SLP-76 protein phosphorylated at Ser376. A SLP-76 rabbit antibody has been coated onto the microwells. After incubation with cell lysates, both phospho- and non-phospho-SLP-76 proteins are captured by the coated antibody. Following extensive washing, a phospho-SLP-76 (Ser376) mouse antibody is added to detect the captured phospho-SLP-76 protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of SLP-76 phosphorylated at Ser376.
Antibodies in this kit are custom formulations specific to kit.
PathScan® Phospho-SLP-76 (Ser376) Sandwich ELISA Kit detects endogenous levels of SLP-76 protein phoshorylated at Ser376 in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is a hematopoietic adapter protein that is important in multiple biochemical signaling pathways and necessary for T cell development and activation (1). ZAP-70 phosphorylates SLP-76 and LAT as a result of TCR ligation. SLP-76 has amino-terminal tyrosine residues followed by a proline rich domain and a carboxy-terminal SH2 domain. Phosphorylation of Tyr113 and Tyr128 result in recruitment of the GEF Vav and the adapter protein Nck (2). TCR ligation also leads to phosphorylation of Tyr145, which mediates an association between SLP-76 and Itk, which is accomplished in part via the proline rich domain of SLP-76 and the SH3 domain of ITK (3). Furthermore, the proline rich domain of SLP-76 binds to the SH3 domains of Grb2-like adapter Gads (3,4). In resting cells, SLP-76 is predominantly in the cytosol. Upon TCR ligation, SLP-76 translocates to the plasma membrane and promotes the assembly of a multi-protein signaling complex that includes Vav, Nck, Itk and PLCγ1 (1). The expression of SLP-76 is tightly regulated; the protein is detected at very early stages of thymocyte development, increases as thymocyte maturation progresses, and is reduced as cells mature to CD4+ CD8+ double-positive thymocytes (5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc.
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|78222C||1 Kit (96 assays)||$489.00.0|