Protein extracts were prepared using lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% IGEPAL CA-630, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM ethylenediaminetetraacetic acid) supplemented with protease and phosphatase inhibitor cocktails (Sigma and Roche). Proteins were immobilized on Immobilon FL PVDF membranes (Millipore) and probed with the following antibodies diluted in Odyssey blocking buffer (LI-COR): anti-PTEN (D4.3) (1:1000, CST), anti-pAkt S473 (1:250, CST), anti-Akt (1:1000, CST), anti-pS6, S235/236 (D57.2.2E) (1:500, CST), anti-S6 (5G10) (1:1000, CST) and anti-tubulin (DM1A) (1:5000, Abcam). Rabbit and mouse IR dye secondary antibodies (LI-COR Biosciences) were used at 1:10 000. Blots were scanned with the Odyssey imaging system and quantified with the Image Studio Lite software (LI-COR).
Rabbit and mouse ir dye secondary antibodies
Manufactured by LI COR
The Rabbit and Mouse IR Dye Secondary Antibodies are fluorescent-labeled secondary antibodies designed for use in immunofluorescence applications. These antibodies are conjugated with near-infrared dyes that can be detected using LI-COR's imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Immobilon fl pvdf membrane, by Merck Group (2 mentions)
Image studio lite software, by LI COR (1 mentions)
Anti tubulin dm1a, by Abcam (1 mentions)
Prism 6, by GraphPad (1 mentions)
Incucyte s3 live cell imaging, by Sartorius (1 mentions)
Anti α tubulin dm1a, by Merck Group (1 mentions)
2 protocols using rabbit and mouse ir dye secondary antibodies
1
Quantitative Western Blot Analysis
2
Murine Prostate Cell Growth Assay with K02288 Inhibitor
Check if the same lab product or an alternative is used in the 5 most similar protocols
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CaP2 and CaP8 murine prostatic cells were provided by Dr. Hong Wu (UCLA) and maintained as described (Jiao et al., 2007 (link)) and confirmed to be mycoplasma negative by PCR. Growth curves of treated cells with indicated amount of K02288 were obtained using phase object confluence as monitored every 2 hr by IncuCyte S3 live cell imaging (Essen Bioscience). Significance between genotype was assessed by one-way ANOVA using Prism 6.0 software (GraphPad). Protein extracts were prepared after 1 hr treatment with K02288 using RIPA lysis buffer (10 mM Tris–HCl (pH 8.0), 140 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulphate, 0.1% deoxycholate, 1 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Roche). Proteins were immobilized on Immobilon FL PVDF membranes (Millipore) and probed with the following antibodies diluted in Odyssey blocking buffer (LI-COR): anti-pAKT S473 (1:500, CST), anti-AKT (1:500, CST), and anti-α-TUBULIN (DM1A) (1:2000, Sigma). Rabbit and mouse IR dye secondary antibodies (LI-COR Biosciences) were used at 1:10 000. Blots were scanned with the Odyssey imaging system (LI-COR).
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