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Akt1 is involved in cellular survival pathways, by inhibiting apoptotic processes. Akt1 is also able to induce protein synthesis pathways, and is therefore a key signalling protein in the cellular pathways that lead to skeletal muscle hypertrophy, and general tissue growth.
Since it can block apoptosis, and thereby promote cell survival, Akt1 has been implicated as a major factor in many types of cancer.
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Phospho-AKT (S473) Polyclonal Antibody (STJ90166)
2. What was used? Primary antibody: Phospho-Akt (S473) Polyclonal antibody (STJ90166) Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot Materials for validation: Mouse liver/colon lysates Click for product data sheet PDF
3. What was the protocol? Treatment of Materials Cells were lysed in 1.5 mls of 1X standard RIPA lysis buffer with protease inhibitors (PMSF and aprotinin) added. After lysis, samples were spun down and 4X sample buffer added. About 35 ul of the lysed cells was separated by SDS-PAGE at 10% and then transferred and blotted Gel Electrophoresis 10% polyacrylamide gel, constant voltage 120V, 80 min. 50 ug per lane. Transfer Nitrocellulose membrane Blocking 1X TBST with 10% skimmed milk powder for 45 minutes. Primary Antibody Application 1:1000 primary antibody was added to the solution of 1X TBST with 2.5% skimmed milk powder, 4?C for overnight. Secondary Antibody Application Secondary antibody (anti-rabbit HRP) was added to the solution of 1X TBST with 2.5% skimmed milk powder with 1:7500 dilution ratio, for 1.5 hours. Visualisation ECL kit
4. What was the protocol? Gel electrophoresis information: 10 % polyacrylamide gel, constant voltage 90 V for 90 min. Transfer information: nitrocellulose membrane, 1 piece of gel (semi-dry transfer) at 260 mA for 3 hours Gel No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band kDa Visualization time 1 Mouse Colon Wild type, no Tx 50 ug Phospho-Akt (S473) antibody (STJ90166) As indicated 1:7500 50 kDa 20min ¨C 1 hour 2 Mouse Colon Wild type, 3% DSS 50 ug 1:7500 50 kDa 20min ¨C 1 hour 3,4 Mouse Colon Rassf1a-/-, no Tx 50 ug Phospho-Akt (S473) antibody (STJ90166) As indicated 1:7500 50 kDa 20min ¨C 1 hour 5, 6 Mouse Colon Rassf1a-/-, 3% DSS 50 ug Phospho-Akt (S473) antibody (STJ90166) As indicated 1:7500 50 kDa 20min ¨C 1 hour 7.8 Mouse liver Rassf1a-/-, 3% DSS + Metformin 50 ug Phospho-Akt (S473) antibody (STJ90166) As indicated 1:7500 50 kDa 20min ¨C 1 hour
5. What were the results?
6. ¡°The results demonstrate that the pS473 Akt can specifically recognize activated Akt in mouse samples. Most likely it will recognize human Akt. Antibody appears quite good.¡± Testing results were provided independently by Professor Shairaz Baksh at the University of Alberta. They stated that: What did the customer think? Antibody Specificity: Antibody Rating:
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