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Akt Vipolnennih Rabot Ks2 Blank Kazahstan

1 Jan 2000admin

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• Western blot analysis of recombinant Akt1, Akt2 and Akt3 proteins, and extracts from HeLa, C2C12, C6 and COS cells, using Akt (pan) (11E7) Rabbit mAb. • Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Akt (pan) (11E7) Rabbit mAb in the presence of control peptide (left) or Akt (pan) (11E7) Blocking Peptide #1085 (right). • Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Akt (pan) (11E7) Rabbit mAb. • Immunohistochemical analysis of paraffin-embedded human melanoma, using Akt (11E7) Rabbit mAb. • Immunohistochemical analysis using Akt (pan) (11E7) Rabbit mAb on SignalSlide(TM) Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)). Note the lack of phosphorylated Akt-associated stain at the membrane of the LY294002 treated cells. • Immunohistochemical analysis of paraffin-embedded HeLa cells untreated (left) or transfected with Akt 1/2/3 Kinases ShortCut® siRNA Mix (New England BioLabs #N2005) (right), using Akt (pan) (11E7) Rabbit mAb (top) or Cleaved Caspase-3 (Asp175) #9661 (bottom).

Note the induction of cleaved caspase-3 in Akt deficient cells. • Confocal immunofluorescent analysis of HeLa cells, serum-starved (left) or insulin-treated (right), using Akt (pan) (11E7) Rabbit mAb (green). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). • Flow cytometric analysis of untreated Jurkat cells, using Akt (pan) (11E7) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

Western Blotting Protocol For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween ® 20 at 4°C with gentle shaking, overnight. NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution. Dr house saison 8 vf torrent. Solutions and Reagents From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: Western Blotting Application Solutions Kit NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

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• 20X Phosphate Buffered Saline (PBS): () To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2O, mix. • 10X Tris Buffered Saline (TBS): () To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH 2O, mix. • 1X SDS Sample Buffer: Blue Loading Pack () or Red Loading Pack () Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.

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Dilute to 1X with dH 2O. • 10X Tris-Glycine SDS Running Buffer: () To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH 2O, mix. • 10X Tris-Glycine Transfer Buffer: () To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH 2O, mix. • 10X Tris Buffered Saline with Tween ® 20 (TBST): () To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH 2O, mix. • Nonfat Dry Milk: (). • Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well. • Wash Buffer: () 1X TBST.

• Bovine Serum Albumin (BSA): (). • Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well. • Biotinylated Protein Ladder Detection Pack: ().

• Prestained Protein Marker, Broad Range (11-190 kDa): (). • Blotting Membrane and Paper: () This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended. • Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (). • Detection Reagent: SignalFire™ ECL Reagent (). Protein Blotting A general protocol for sample preparation. • Treat cells by adding fresh media containing regulator for desired time.

• Aspirate media from cultures; wash cells with 1X PBS; aspirate. • Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. • Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). • Heat a 20 µl sample to 95–100°C for 5 min; cool on ice. • Microcentrifuge for 5 min. • Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).