磷酸- 4E- BP1（Thr37/46）（236B4）兔单克隆抗体 Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb

Applications Key:  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by Western blot.

Protocols

2846:

Flow

Specificity / Sensitivity

Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor^® 488 Conjugate) detects endogenous levels of 4E-BP1 only when phosphorylated at Thr37 and/or Thr46. This antibody may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr37 and Thr46 of mouse 4E-BP1. The antibody was conjugated to Alexa Fluor^® 488 under optimal conditions with an F/P ratio of 2-5.

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (green) or LY294002, Wortmannin and U0126-treated (blue), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor^® 488 Conjugate).

Description

Cell Signaling Technology antibody conjugated to Alexa Fluor^® 488 fluorescent dye and tested in-house for direct flow cytometric analysis of human cells. The unconjugated antibody, #2855, reacts with Phospho-4E-BP1 (Thr37/46) from human, mouse, rat and monkey. CST expects that phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor^® 488 Conjugate) will also recognize Phospho-4E-BP1 in these species.

磷酸- 4E- BP1（Thr37/46）（236B4）兔单克隆抗体 Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb

A. Solutions and Reagents

1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄ and 0.24 g KH₂PO₄ in 800 mL distilled water (dH₂O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
2. Formaldehyde (methanol free).
3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS. Store at 4°C.

B. Fixation

1. Collect cells by centrifugation and aspirate supernatant.
2. Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
3. Fix for 10 minutes at 37°C.
4. Chill tubes on ice for 1 minute.
5. For extracellular staining with antibodies that do not require permeabilization, proceed to step D1 or store cells in PBS with 0.1% Sodium Azide at 4°C; for intracellular staining, proceed to permeabilization step C1.

C. Permeabilization

1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, to remove fix prior to permeabilization, pellet cells by centrifugation and resuspend in 90% methanol.
2. Incubate 30 minutes on ice.
3. Proceed with staining or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.

1. Aliquot 0.5-1x10⁶ cells into each assay tube (by volume).
2. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
3. Resuspend cells in 100 µl Incubation Buffer per assay tube.
4. Block in Incubation Buffer for 10 minutes at room temperature.
5. Add the unconjugated, biotinylated, or fluorochrome-conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the appropriate dilution).
6. Incubate for 1 hour at room temperature.
7. Rinse as before in Incubation Buffer by centrifugation.
8. If using a fluorochrome-conjugated primary antibody, resuspend cells in 0.5 ml PBS and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to step D9.
9. Resuspend cells in fluorochrome-conjugated secondary antibody* or fluorochrome-conjugated avidin, diluted in Incubation Buffer at the recommended dilution.
10. Incubate for 30 minutes at room temperature.
11. Rinse as before in Incubation Buffer by centrifugation.
12. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step E1.

E. Optional DNA Stain

1. Resuspend cells in 0.5 ml of DNA dye (e.g. DRAQ5^® #4084).
2. Incubate for at least 5 mins at room temperature.
3. Analyze cells in DNA stain on flow cytometer.