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Forkhead box protein 03 also known as FOXO3 is a transcriptional activator encoded by the FOXO3 gene in humans. The forkhead family of transcription factors are known for their fork head DNA binding domains. FOXO3 is thought to cause apoptosis in the absence of survival factors in cells. The Phospho-FoxO3A (S253) Polyclonal Antibody detects endogenous levels of FoxO3A protein only when phosphorylated at S253.
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Phospho-FoxO3A (S253) Polyclonal Antibody (STJ90275)
2. What was used? Primary antibody: STJ90275 Phospho-FoxO3A (S253) Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot Materials for validation: MCF7 cell line Click for product data sheet PDF
3. What Was The Protocol? Treatment of Materials When cells were cultured to 70%-80% plating density, lysis buffer with protease inhibitors (Sigma) and equal volume of 2X loading buffer were added for 10 minutes at 95?C. Gel Electrophoresis 10% Polyacrylamide gel with a constant voltage of 160V for 60 minutes. Transfer 0.45 um PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100V for 75 minutes. Blocking 1X TBST with 5% BSA for 1 hour. Primary Antibody Probing Primary antibody was added to the solution of 1X TBST with 5% BSA, and left at 4?C overnight. Membrane Wash Membrane was washed with 1X TBST 3 times. Secondary Antibody Probing Secondary antibody was added to the solution of 1X TBST with 5% BSA with a 1:2500 dilution ratio, for 1 hour. Membrane Wash Membrane was washed with 1X TBST 3 times. Visualization ECL visualization for 3 min (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).
4. What was the protocol? Gel electrophoresis information: 10% polyacrylamide gel, constant voltage of 160V for 60 minutes. Transfer information: 0.45 um PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100V for 75 minutes. No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualization time 1 MCF7 15ug STJ90275 Phospho- FoxO3A (S253) antibody 1:1000 1:2500 ~70KD 30S
5. What were the results?
6. What did the customer think? Antibody Specificity: Antibody RPPA Rating: Testing results were provided independently by R. Bernardo at the Royal College of Surgeons in Ireland. They stated ¡°In MCF7 cell line, there were target bands detected by FoxO3A (S253) antibody STJ90275 Antibody.¡±
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