Protein levels in cells were measured using Western blot assay. The transfected cells were lysed in Triton lysis buffer (Solarbio, Beijing, P.R. China) for 30 min on ice, and the protein concentration was determined by Bicinchoninic Acid (BCA) Kit (Solarbio). The protein was resolved over 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (20 (link)). The membrane was blocked in blocking buffer (5% nonfat milk) for 2 h at room temperature, and then incubated with primary antibodies against p-Akt (1:1,000), Akt (1:1,000), Bcl-2 (1:1,000), Bax (1:1,000), p27 (1:1,000), p21 (1:1,000), procaspase 3 (1:1,000), cleaved caspase 3 (1:1,000), and actin (1:2,000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. A secondary antibody (1:2,000) (Cell Signaling Technology, Danvers, MA, USA) was used for 1 h at room temperature. The bands were detected by chemiluminescence and autoradiography using an X-ray film (Applygen, Beijing, P.R. China), and densitometric measurements were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
X ray film
Manufactured by Applygen
Sourced in China
X-ray film is a type of photographic film used in medical and industrial imaging applications. It is designed to capture and record images produced by X-ray radiation passing through the body or an object. The film is sensitive to X-rays and, when exposed, it produces a latent image that can be developed and viewed.
Automatically generated - may contain errors
Lab products found in correlation
Actin, by Santa Cruz Biotechnology (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Triton lysis buffer, by Solarbio (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bca kit, by Solarbio (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Santa Cruz Biotechnology (1 mentions)
Procaspase 3, by Santa Cruz Biotechnology (1 mentions)
Sc 271759, by Santa Cruz Biotechnology (1 mentions)
Image labtm software, by Bio-Rad (1 mentions)
3 protocols using x ray film
1
Western Blot Analysis of Protein Levels
2
Protein Expression Analysis in HMEC-1 Cells
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HMEC-1 cells were lysed by 1% Triton X-100 and 1 mM PMSF (pH 7.4) over ice for 30 min for protein extraction. Purity and concentration of protein in the supernatant of the whole-cell extracts were qualified by the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Protein (0.1 mg) was separated by SDS-PAGE and was transferred onto a PVDF membrane. The membranes were blocked in 5% non-fat dry milk/0.05% Tween for 1 h at room temperature, after which the blots were probed by primary antibodies overnight at 4°C for the detection of VEGF (orb303954, Biorbyt, San Francisco, CA, USA), VEGFR2 (orb186489), CD144 (orb375049), eNOS (orb29800), Bcl-2 (orb10173), Bax (orb378567), caspase-3 (sc-271759, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (sc-47724), caspase-9 (ab25758, Abcam, Cambridge, MA, USA), p-PI3K (ab182651), PI3K (ab191606), p-AKT (ab38449), AKT (ab8805), p-Jak1 (ab215338), Jak1 (ab47435), p-Tyk2 (ab138394), Tyk2 (ab223733), p-Stat1 (ab30645), Stat1 (ab31369), p-Stat2 (ab53132), and Stat2 (ab32367). The membranes were then incubated with the secondary antibodies for 1 h at room temperature. After rinsing, the positive bands were visualized by chemiluminescence and autoradiography using X-ray film (Applygen Technologies Inc., Beijing, China). Intensity of bands was quantified using Image Lab TM Software (Bio-Rad, CA, USA).
3
GH Stimulation of Osteoblast Proteins
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The osteoblasts (grown to near 70% confluent) were treated with 50 µg /mL GH for 48 h and then lysed over ice with Triton X-100 (1%) and PMSF (1 mmol/L, pH 7.4) for 30 min. The collected lysate was then centrifuged at 14,000× g for 15 min at 4 °C, while the separated supernatant (i.e., total cell protein) was used in the Western blot analysis or immediately stored at −80 °C until analysis. A BCA Protein Assay Kit was used to determine the protein concentration of the supernatant. A protein sample of 0.1 mg was resolved over SDS-PAGE (10–12%) and transferred to a polyvinylidene fluoride membrane. The protein blot was blocked with a blocking buffer (containing 5% nonfat dry milk and 0.05% Tween 20 in 20 mmol/L TBS, at pH 7.6) at 20 °C for 60 min, incubated with monoclonal antibody in blocking buffer for 240 min at 4 °C, incubated with secondary antibody horseradish peroxidase conjugate and then detected via chemiluminescence and autoradiography using X-ray film (Applygen Technologies Inc., Beijing, China). Afterward, LabWorks 4.5 image analysis software (UVP Bioimaging System, Upland, CA, USA) was used to perform the densitometric measurements of the bands, while endogenous standard β-actin was employed in this evaluation to normalize the band density values of the targeted protein blots.
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