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Immunofluorescence (Immunocytochemistry)

Immunofluorescent analysis of MCF7 cells, serum-starved (left) or insulin-treated (right), using PathScan® Signaling Nodes Multiplex IF Kit. Red = Phospho-Akt (Ser473), green = Phospho-p44/42 (Thr202/Tyr204), and blue pseudocolor = Phospho-S6 (Ser235/236).

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Immunofluorescence (Immunocytochemistry)

Immunofluorescent analysis of insulin-treated MCF7 cells (human breast adenocarcinoma), following pretreatment with kinase specific inhibitors LY294002 (PI3 Kinase Inhibitor) #9901 or U0126 (MEK1/2 Inhibitor) #9903 for the indicated times.

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde, 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh, store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100):
    To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100
  4. Antibody Dilution Buffer(1X PBS / 1% BSA / 0.3% Triton™ X-100):
    To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed, and stained directly in multi-well plates, chamber slides, or on coverslips.

  1. Aspirate liquid, and then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
    NOTE: Formaldehyde is toxic, use only in fume hood.
  2. Allow cells to fix for 15 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid, light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary cocktail.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Prepare detection cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  7. Incubate 1–2 hours at room temperature in the dark.
  8. Rinse three times in 1X PBS for 5 minutes each.
  9. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), or Prolong® Gold Antifade Reagent with DAPI (#8961).
  10. For best results examine specimens immediately using appropriate excitation wavelengths. For long-term storage, store slides at 4°C protected from light.

posted July 2010

revised August 2011

protocol id: 424

Kit Includes

Products Included No. Volume Applicaton Dilution Reactivity
Primary Cocktail 8998 100 µl Immunofluorescence (Immunocytochemistry) 1:100 Human
Detection Cocktail 8997 100 µl Immunofluorescence (Immunocytochemistry) 1:100 N/A
Kit Analytes Detection Dye Ex(max) (nm) Em(max) (nm)
Phospho-Akt (Ser473) Alexa Fluor® 555 555 565
Phospho-p44/42 Erk1/2 (Thr202/Tyr204) Alexa Fluor® 488 495 519
Phospho-S6 Ribosomal Protein (Ser235/236) Alexa Fluor® 647 650 665

Product Description

Traditional biochemical and lysate-based assays (e.g., western blot, immunoprecipitation, ELISA) have been integral in the analysis of individual signaling events, however they are limited in their ability to monitor the phosphorylation and subcellular localization of multiple proteins on a per cell basis. PathScan® Signaling Nodes Multiplex IF Kit offers a novel method to simultaneously monitor signaling through key pathway nodes using manual immunofluorescence microscopy, or automated imaging and laser scanning high content platforms. These kits contains a cocktail of three high quality primary antibodies targeted against phospho-Akt (Ser473), phospho-p44/42 (Thr202/Tyr204), and phospho-S6 (Ser235/236) and a detection cocktail utilizing the Alexa Fluor® series of fluorescent dyes. Antibody formulation and dye pairings have been pre-optimized and each kit contains enough reagents for 100 assays (based on a working volume of 100 μL/test).

Specificity / Sensitivity

Phospho-Akt (Ser473) antibody detects endogenous levels of Akt only when phosphorylated at Ser473. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody detects endogenous levels of p44 and p42 MAP kinase (Erk1 and Erk2) when dually phosphorylated at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2), and singly phosphorylated at Thr202. This antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinases. Phospho-S6 ribosomal protein (Ser235/236) antibody detects endogenous levels of ribosomal protein S6 only when phosphorylated at Ser235 and Ser236.

Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser473 of human Akt, Thr202/Tyr204 of human p44 MAP kinase, and Ser235/Ser236 of human ribosomal protein S6.

Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis (1-3). This protein kinase is a downstream effector of phosphoinositide-3 kinase (PI3K), and is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4), as well as by phosphorylation within the carboxy terminus at Ser473 by the mTOR-rictor complex (TORC2) (5). This pathway is down-regulated following dephosphorylation of phosphatidyl-inositol 3,4,5 triphosphate by PTEN, as well as by deactivation of PI3K with targeted small molecule inhibitors such as wortmannin and LY294002 (2,3,6,7).

p70 S6 kinase, a mitogen activated Ser/Thr protein kinase downstream of PI3K and the mTOR-raptor complex (mTORC1), phosphorylates the S6 protein of the 40S ribosomal subunit leading to an increase in translation of mRNA transcripts that contain an oligopyrimidine tract in their 5’ untranslated region (8). These particular mRNA transcripts (5’TOP) encode proteins involved in cell cycle progression, as well as ribosomal proteins and elongation factors necessary for translation (8,9). Important S6 ribosomal protein phosphorylation sites include several residues (Ser235, Ser236, Ser240 and Ser244) located within a small, carboxy-terminal region of the S6 protein (10,11).

Both p44 and p42 mitogen-activated protein (MAP) kinases (Erk1 and Erk2, respectively) play a critical role in the regulation of cell growth and differentiation (12-15). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones, and neurotransmitters. Activation of MAP kinases occur through phosphorylation of Thr202/Tyr204 on human Erk1 and Thr185/Tyr187 on human Erk2 at the sequence T*EY* by a pair of upstream MAP kinase kinases (MEK1/2) (16,17). Erk proteins are negatively regulated by a family of dual specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (18), along with MEK inhibitors such as U0126 and PD98059. Erk dependent phosphorylation of TSC2 at Ser663 leads to the functional inactivation of the TSC1/TSC2 inhibitory complex, and subsequent downstream activation of S6 ribosomal protein through the mTORC1/p70 S6K signaling cascade (19).

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Entrez-Gene Id 207 , 208 , 10000 , 5595 , 5594 , 6194
Swiss-Prot Acc. P31749 , P31751 , Q9Y243 , P27361 , P28482 , P62753

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