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7179
PathScan® Total 4E-BP1 Sandwich ELISA Kit
ELISA Kits
ELISA Kit

PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179

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Figure 1: Treatment of HEK-293T cells with amino acids and insulin stimulates phosphorylation of 4E-BP1 at Thr37/46, detected by PathScan® Phospho-4E-BP1 (Thr37/46) Sandwich ELISA Kit #7216, but does not affect the level of total 4E-BP1 protein detected by PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179. HEK-293T cells (70-80% confluent) were starved overnight and deprived of amino acids for 1 hour. The amino acids were replenished for 1 hour. Cells were either untreated or stimulated with 100 nM insulin for 30 minutes at 37ºC. λ phosphatase treatment of control cell lysates (4000 U/mL for 60 minutes at 37ºC) abolishes the basal phosphorylation of 4E-BP1 as shown by both sandwich ELISA and Western analysis. The absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots, using 4E-BP1 Antibody #9452 (left panel) or Phospho-4E-BP1 (Thr37/46) Antibody #9459 (right panel), are shown in the bottom figure.
Figure 2: The relationship between the protein concentration of the lysate from amino acid (AA)/untreated and AA/insulin-treated HEK-293T cells and the absorbance at 450 nm is shown.
Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from HEK-293T cells and incubated in microwells coated with the 4E-BP1 capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of HEK-293T cell starting lysates (lanes 1 & 2) and the captured protein (lanes 3 & 4) was performed using 4E-BP1 Antibody #9452. The major band detected in the captured material (lanes 3 & 4) corresponds to 4E-BP1.
To Purchase # 7179
Cat. # Size Qty. Price
7179C
1 Kit  (96 assays)

Important Ordering Details

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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
4E-BP1 Rabbit mAb Coated Microwells 96 tests
4E-BP-1 Mouse Detection mAb 1 ea Green (Lyophilized)
Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated) 1 ea Red (Lyophilized)
Detection Antibody Diluent 2 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

CST's PathScan® Total 4E-BP1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of 4E-BP1. A 4E-BP1 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, 4E-BP1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a 4E-BP1 Mouse Detection Antibody is added to detect the captured 4E-BP1 protein. Anti-mouse IgG, HRP-linked Antibody #7076 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 4E-BP1.

*Antibodies in 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

CST's PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179 detects endogenous levels of 4E-BP1. As shown in Figure 1, using CST's PathScan® Phospho-4E-BP1 (Thr37/46) Sandwich ELISA Kit #7216, a significant induction of 4E-BP1 phosphorylation at Thr37/46 is detected in serum and amino acid starved HEK-293T cells treated with insulin for 30 minutes after replenishing the amino acids. The level of total 4E-BP1 (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179.
This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Background

Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).

Pathways

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