Revision 9

#78222

Store at +4C

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

1 x 96 tests

Species Cross Reactivity:
H

UniProt ID:
#Q13094

Entrez-Gene Id:
#3937

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

Web:

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Color Storage Temp
SLP-76 Rabbit mAb Coated Microwells, 384 Well ("D" Kit Only)69110384 tests+4C
SLP-76 Rabbit mAb Coated Microwells4364296 tests+4C
Phospho-SLP-76 (Ser376) Mouse Detection mAb701641 eaGreen (Lyophilized)+4C
Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)133041 eaRed (Lyophilized)+4C
Detection Antibody Diluent1333911 mlGreen+4C
HRP Diluent1351511 mlRed+4C
TMB Substrate700411 ml+4C
STOP Solution700211 ml+4C
Sealing Tape545032 ea+4C
ELISA Wash Buffer (20X)980125 ml+4C
ELISA Sample Diluent1108325 mlBlue+4C
Cell Lysis Buffer (10X)980315 ml-20C

Kit contents scale proportionally with size, except sealing tape.
Example: The V1 kit contains 5X the listed quantities above, but will exclude the sealing tape.

For the “C” and “V” kits, the supplied 96-well strip plate consists of twelve 8-well strips in a support frame. This enables custom plate configurations.

For the “D” kits, the supplied 384-well microplate is a single piece of material and does not break apart.

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.

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. This 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).

Background References

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PathScan is a registered trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 9

#78222

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

Cell Signaling Technology Logo

PathScan® Sandwich ELISA Protocol

(Colorimetric 96 well)

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 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 1 mL of reconstituted Detection Antibody to 10 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 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 1 mL volume of reconstituted HRP-Linked Antibody to 10 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.
  5. HRP Diluent: Red colored diluent for reconstitution and dilution of the HRP‑Linked Antibody.
  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: When using to prepare cell lysates, add Protease/Phosphatase Inhibitor Cocktail (100X) (#5872, not supplied) and 1 mM Phenylmethanesulfonyl Fluoride (PMSF, #8553, not supplied) 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 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 the top of the 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 every well.
    3. After each wash, strike plates on clean paper 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 (Step 4 above).
  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 (Step 4 above).
  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 sec.

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: Clean the underside of all wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP Solution.

PathScan® Sandwich ELISA Protocol

(Colorimetric 384 well)

A. Solutions and Reagents

NOTE: Prepare solutions with deionized/purified water or equivalent.

  1. 384 Well Microplate: Before opening the sealed bag, allow it to reach room temperature.
  2. Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 1 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 mL of reconstituted Detection Antibody to 10 mL of Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C, although there may be some loss of signal compared to freshly reconstituted antibody.
  3. HRP-Linked Antibody*: Supplied lyophilized as a red colored cake or powder. Add 1 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 mL volume of reconstituted HRP-Linked Antibody to 10 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) to 1X in deionized 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: When using to prepare cell lysates, add Protease/Phosphatase Inhibitor Cocktail (100X) (#5872, not supplied) and 1 mM Phenylmethanesulfonyl Fluoride (PMSF, #8553, not supplied) immediately before use.
  9. TMB Substrate #7004: Bring to room temperature before use.
  10. STOP Solution #7002: Bring to room temperature before use.

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, add 0.5 mL ice-cold 1X Cell Lysis Buffer including 1 mM PMSF and Protease/Phosphatase Inhibitor Cocktail 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 mL of 1X Cell Lysis Buffer including 1 mM PMSF and Protease/Phosphatase Inhibitor Cocktail.
  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

NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.

  1. Prepare all reagents as indicated above (Section A).
  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 results across a range of lysate concentration points.
  3. Add 25 μL of each undiluted or diluted cell lysate to the appropriate wells. Seal with tape and press firmly onto the top of the 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, 100 μl each time for every well.
    3. After each wash, strike plates on clean paper 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 25 μL of 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 (Step 4 above).
  7. Add 25 μL of 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 (Step 4 above).
  9. Add 25 μL of TMB Substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 15 min at room temperature on a plate shaker (400 rpm, moderate agitation).
  10. Add 25 μL of STOP Solution to each well. Shake gently for a few sec.

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: Clean the underside of all wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP Solution.

For Research Use Only. Not for Use in Diagnostic Procedures.

posted August 2025

Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.