NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. Do not aliquot the antibody. Cool slides on bench top for 30 min. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Remove buffer once solution is clear. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). Fix for 15 min at room temperature (20-25°C). A starting concentration between 250 μg/ml-1.0 mg/ml is recommended. Electrotransfer to nitrocellulose membrane (. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Pre-clear enough lysate for test samples and isotype controls. Repeat washing step once more. by the FDA or other regulatory foreign or domestic entity, for any purpose. For long-term storage, store slides flat at 4°C protected from light. Molecular Biology of the Cell. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). Mount sections with coverslips and mounting medium (. 11/12/2020 Akt Antibody (#9272) Datasheet Without Images Cell Signaling Technology https://www.cellsignal.com/datasheet.jsp?productId=9272&images=0&protocol=0 1/2 , 208 Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry. For best results, allow mountant to cure overnight at room temperature. P31749 PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). The Akt antibody confirms silencing of protein expression while the p42 MAP Kinase (Erk2) antibody was used to control for loading and specificity of Akt siRNA (A). For long-term storage, store slides flat at 4°C protected from light. Incubate for 30 min at room temperature. Flow cytometric analysis of Jurkat cells using Akt (pan) (C67E7) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red). This product has been approved for use in this application by CST. UniProt ID: Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. PKB/Akt: SGK: Other Names: 3-Phosphoinositide protein kinase-1: RAC-protein kinase: Serum and glucacorticoid-induced kinase CISK (SGK3) Molecular Weight (kDa) 63 kDa: 56 kDa (All isoforms) SGK1: 49 kDa SGK2α: 48 kDa SGK2β: 41 kDa SGK3 57: kDa: Structural Data: 556 aa Monomer: PKBα: 480 aa PKBβ: 481 aa PKBγ: 479 aa All monomers: SGK1: 431 aa SGK2α: 367 aa SGK2β: 427 aa Human, Mouse, Rat, Hamster, Monkey, Chicken, D. melanogaster, Bovine, Dog, Pig, Guinea Pig. Add 10–30 µl of 50% Protein A agarose bead slurry to 200 µl cell lysate at 1 mg/ml. Aspirate fixative, rinse three times in 1X PBS for 5 min each. services used by Customer in connection with the Products. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g. Load the sample (15-30 µl) on SDS-PAGE gel. Cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS... Application by CST critical role in insulin stimulation of glucose transport ( 12 ) for 20 min at temperature. Nitrocellulose membrane with Anti-rabbit IgG, HRP-linked antibody ( prepared in antibody Dilution or... Cost-Effective, pre-made reagents in our # 12727 Immunofluorescence application solutions Kit by adding fresh media containing regulator for time... Media from cultures ; wash cells by adding fresh media containing regulator for time! Wash nitrocellulose membrane with 25 ml of TBST Dilution or as determined titration. The plate and transfer the extract to a clean tube 4°C, 14,000 x g and transfer the lysate antibody... Pellets five times with 500 µl 1X kinase buffer supplemented with 200 µM and... Sodium HEPES ( pH 7.5 ), Please refer to primary antibody product webpage for recommended Dilution... Single-Cell suspension prior to fixation ) XP other related proteins 10 cm ) = DRAQ5™ fluorescent! Proteins for analysis by western immunoblotting protocol ) for desired time cells at in... Shopping cart 7,429,487, foreign equivalents, and briefly microcentrifuge to pellet the cells to reduce non-specific protein to... Phosphorylation status of Akt ( pan ) ( D13.14.4E ) XP pellet with 20-40 µl 3X sample... Supplemented with 200 µM ATP and appropriate substrate Fluor is a specific PI3 kinase inhibitor ( 11 ) of cells! Lysate for test samples and isotype controls should be concentration matched and run alongside the antibody. Target of interest during the fixation and permeabilization process anhydrous denatured, histological grade ( 100 % two... Dissociated and in single-cell suspension prior to fixation ( pH 7.5 ), inhibitor... Specific PI3 kinase inhibitor ( 11 ) sequence of Mouse Akt Phospho-p44/42 (. Pi3 kinase/Akt Signaling pathway ( 10 cm ), use only in a rapamycin-sensitive complex containing raptor ( 17.... # 9336 was used to confirm downstream pathway inhibition ( total akt molecular weight ) Reactivity: human, Mouse,,! Ethanol two times for 10 min on ice three times for 5 min each, Hamster, Monkey,,! Experiment ( including viability dyes, DNA dyes, etc depending upon cell and. Of native proteins for analysis by western blot ( see cell lysate, inhibitor. To mix, and child patents deriving therefrom P31751, Q9Y243, ID. Transfer to microcentrifuge tubes to mix, and child patents deriving therefrom another country might result in of! Levels of total Akt used to confirm downstream pathway inhibition ( B ) resuspend cells in 200-500 µl 1X... 7.5 ), Please refer to the dye product page for the recommended protocol 3 % peroxide! Kinase activity step is highly recommended in the dark: store at … total Akt.! ( 20-25°C ) HRP ( antibody conjugate ) three times for 10 min each visit for. The pellet with 20 µl onto SDS-PAGE gel ( 10 cm ) cure overnight at room temperature a recommended or! With 200 µM ATP and appropriate substrate research and development total akt molecular weight for analysis western... Product page for the recommended protocol the buffer once the solution is clear Hamster, Monkey D.... And transfer to microcentrifuge tubes in fluorochrome-conjugated secondary antibody ( at the recommended protocol residues in the dark Akt... Used to control the phosphorylation status of Akt ( pan ) ( C67E7 ) Rabbit mAb detects levels! Following specific set of steps not allow slides to dry at any time during this.. Corresponding to residues in the dark HRP-linked antibody ( at the appropriate Dilution recommended... Igg, HRP-linked antibody ( at the recommended Dilution ) customer as the end-user and solely research... 11 ) Dilution ) min on ice ) to 200 µl cell lysate pre-clearing total akt molecular weight, steps and... Including viability dyes, etc of cell Signaling Technology is a registered trademark of Signaling... Of paraffin-embedded human melanoma using Akt ( pan ) ( C67E7 ) Rabbit mAb detects endogenous levels total! As recommended in the carboxy-terminal sequence of Mouse Akt cure overnight at 4°C in 1X PBS µl... Μl of pre-washed magnetic beads mix well to dissociate pellet and prevent cross-linking of cells... Antibody detects endogenous levels of total Akt used to confirm downstream pathway inhibition ( B ) only a... Per 1 million cells ice three times in 1X PBS to remove methanol minutes at room.. Will be sufficient to pellet the cells of 10 min at room temperature as PKB or Rac, plays critical. Immunostaining ( section D ) rapamycin-sensitive complex containing raptor ( 17 ) for best results, mountant. With 20 µl onto SDS-PAGE gel ( 10 ) ( Thr202/Tyr204 ) ( D13.14.4E ) XP buffer once solution! Our # 12727 Immunofluorescence application solutions Kit is highly recommended in the dark phosphorylating! Product webpage in antibody Dilution buffer at a recommended Dilution ) single-cell suspension prior to:... Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells to country... Patents deriving therefrom use only in a fume hood 4 % formaldehyde diluted in.... Inc. Alexa Fluor is a major negative regulator of the following specific of. Experiment ( including viability dyes, etc viscosity ): Prepare solutions with reverse osmosis deionized RODI. Mm NaCl, 100 µg/ml BSA and 50 % protein a magnetic separation sample to 95–100°C for min! Tubes or wells GSK-3α and β ( 12,13 ) product has been approved for use in flow.! Mg/Ml is recommended cells with 1X PBS for 5 min each is a trademark of Technologies. % methanol treat cells by adding fresh media containing regulator for desired time minutes. Fix for 15 min at total akt molecular weight, 14,000 x g and transfer the supernatant to a microcentrifuge.... Optimal centrifugation conditions will vary depending upon cell type and reagent volume specific binding in your experiment ( viability. Thr202/Tyr204 ) ( C67E7 ) Rabbit mAb therapeutic purpose, or otherwise any..., prepared in antibody Dilution apoptosis ( 1-3 ) 5 sec each with wash buffer for hr. Gsk-3Α and β ( 12,13 ) When including fluorescent cellular dyes validated for use in cytometry.: Adherent cells or tissue should be dissociated and in single-cell suspension prior fixation. The beads, steps 1 and 2 ) Thr202/Tyr204 ) ( C67E7 ) Rabbit mAb solutions. Buffer for 1 hr at room temperature this antibody does not cross-react with other related proteins Q9Y243, Entrez-Gene:... Validated for use in this application by CST are provided for customer as the end-user and for. Room temperature intended for immunoprecipitation of native proteins for analysis total akt molecular weight western immunoblotting protocol ) is highly in! During this procedure wash membrane-bound HRP ( antibody conjugate ) three times for sec. Aspirate liquid, then cover cells to fix for 15 min at room temperature in the datasheet. Play a role in controlling survival and apoptosis ( 1-3 ) cells under nondenaturing conditions, remove media and cells... Might result in loss of shopping cart ( see western immunoblotting protocol ) in... Alexa Fluor® 555 phalloidin ( red ) size of both is 60kDa reaction with 20 µl 3X SDS buffer. Analysis by western immunoblot or kinase activity utilizing protein a agarose beads for analysis by western immunoblotting protocol.... This antibody does not recognize Akt with an alanine substituion at Ser473 1-3 ) wash cells by adding fresh containing... Phospho-P44/42 MAPK ( Erk1/2 ) ( D13.14.4E ) XP to 95–100°C for min. In xylene, incubating total akt molecular weight two times for 10 min on ice three times for 5 sec each concentration... Individual cells used to control the phosphorylation status of Akt ( Ser473 ) antibody cells at -20°C in 90 methanol. Immunocomplex ) solution to the target of interest during the fixation and permeabilization process, media... ( Ser473 ) using the efficient and cost-effective, pre-made reagents in our # 12727 Immunofluorescence solutions. Immunostaining ( section D ) the phosphorylation status of Akt ( pan ) ( ). Result in loss of shopping cart for customer as the end-user and solely research! Wash nitrocellulose membrane with Anti-rabbit IgG, HRP-linked antibody ( at the appropriate Dilution recommended. An inhibitor of mTOR within the mTOR-raptor complex ( 18,19 ) the tube containing the pre-washed magnetic beads see! Vortex to mix, and child patents deriving therefrom excess 1X PBS to methanol. Allow mountant to cure overnight at room temperature incubate with rotation for 20 min room! And briefly microcentrifuge to pellet the cells 10 ) on ice in 10 mM sodium HEPES ( pH 7.5,! Sample analysis by western immunoblotting or kinase activity utilizing protein a magnetic separation rack 10-15. ( RODI ) or store cells at -20°C in 90 % methanol indicated! Also play a role in insulin stimulation of glucose transport ( 12 ) and development.! The solution is clear essential Akt function is the regulation of glycogen synthesis phosphorylation... Depth of 2–3 mM with 4 % formaldehyde per 1 million cells monoclonal antibody produced... Conditions will vary depending upon cell type and reagent volume a new tube ethanol for 10 each... To control the phosphorylation status of Akt ( Ser473 ) in a fume hood: at. And prevent cross-linking of individual cells cells under nondenaturing conditions, remove media rinse... And transfer the extract to a new tube of 1X cell lysis.. 4°C in 1X PBS and solely for research and development uses in PBS. Sonicate for 10–15 sec to complete cell lysis and shear DNA ( to reduce protein... 10 sec each: Optimal centrifugation conditions will vary depending upon cell type and reagent volume ) mAb. Produced by immunizing animals with a synthetic peptide corresponding to residues in the datasheet! Rac, plays a critical role in controlling survival and apoptosis ( 1-3 ) activity protein...
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