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78222
PathScan® Phospho-SLP-76 (Ser376) Sandwich ELISA Kit 
ELISA Kits
ELISA Kit

PathScan® Phospho-SLP-76 (Ser376) Sandwich ELISA Kit  #78222

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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.
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).
To Purchase # 78222
Cat. # Size Qty. Price
78222C
1 Kit  (96 assays)

Important Ordering Details

Custom Ordering Details:

When ordering five or more kits, please contact us for processing time and pricing.

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Supporting Data

REACTIVITY H

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected
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) 9801 25 ml
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) 9803 15 ml

Product Description

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.

Protocol

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ELISA Colorimetric (Lyophilized)

A. Solutions and Reagents

NOTE: Prepare solutions with purified water.

  1. Microwell strips: Bring all to room temperature before use.
  2. Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 1.0 ml of Detection Antibody Diluent (green solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted Detection Antibody to 10.0 ml of Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.
  3. HRP-Linked Antibody*: Supplied lyophilized as a red colored cake or powder. Add 1.0 ml of HRP Diluent (red solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted HRP-Linked Antibody to 10.0 ml of HRP Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.
  4. Detection Antibody Diluent: Green colored diluent for reconstitution and dilution of the detection antibody (11 ml provided).
  5. HRP Diluent: Red colored diluent for reconstitution and dilution of the HRP‑Linked Antibody (11 ml provided).
  6. Sample Diluent: Blue colored diluent provided for dilution of cell lysates.
  7. 1X Wash Buffer: Prepare by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in purified water.
  8. Cell Lysis Buffer: 10X Cell Lysis Buffer #9803: This buffer can be stored at 4°C for short-term use (1–2 weeks). Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) immediately before use.
  9. TMB Substrate (#7004).
  10. STOP Solution (#7002).

*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.

B. Preparing Cell Lysates

For adherent cells.

  1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
  4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  5. Sonicate lysates on ice.
  6. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
  3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X Cell Lysis Buffer plus 1 mM PMSF.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

C. Test Procedure

  1. After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed and stored at 4°C immediately.
  2. Cell lysates can be undiluted or diluted with Sample Diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets for each kit provide a sensitivity curve that serves as a reference for selection of an appropriate starting lysate concentration. The sensitivity curve shows typical kit assay results across a range of lysate concentration points.
  3. Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C.
  4. Gently remove the tape and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash 4 times with 1X Wash Buffer, 200 µl each time for each well.
    3. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
    4. Clean the underside of all wells with a lint-free tissue.
  5. Add 100 µl of reconstituted Detection Antibody (green color) to each well (refer to Section A, Step 2). Seal with tape and incubate the plate at 37°C for 1 hr.
  6. Repeat wash procedure (Section C, Step 4).
  7. Add 100 µl of reconstituted HRP-Linked secondary antibody (red color) to each well (refer to Section A, Step 3). Seal with tape and incubate the plate for 30 min at 37°C.
  8. Repeat wash procedure (Section C, Step 4).
  9. Add 100 µl of TMB Substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C.
  10. Add 100 µl of STOP Solution to each well. Shake gently for a few seconds.

NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

  1. Read results.
    1. Visual Determination: Read within 30 min after adding STOP Solution.
    2. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP Solution.

posted November 2013

Protocol Id: 204

Specificity / Sensitivity

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.

Background

SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is a hematopoietic adaptor 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 adaptor 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 adaptor 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).

Pathways

Explore pathways related to this product.

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