We analyzed the expression of insulin-like growth factor (IGF)-1 (a stimulator of hair growth) and transforming growth factor (TGF)-β1 (an inhibitor of hair growth) in the four groups of experimental mice. The levels of IGF-1 and TGF-β1 protein in skin were measured by Western blotting. Fifty milligrams of tissue with lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 2 nM EDTA, 1 mM EGTA, 1% Triton X-100) was homogenized (Tissue Tearor, Biospec, Korea) and the homogenized samples (50 μg of protein) were subjected to 15% SDS-PAGE and then transferred to a PVDF membrane. Membranes were blocked in 5% skim milk for 1 h, incubated with primary antibodies (IGF-1, TGF-β1, Abcam, Cambridge, UK) overnight (4 °C) at appropriate dilutions (1:1000), and then incubated with the secondary antibody (goat anti-rabbit IgG, Stressgen, USA) conjugates for 1 h at room temperature. Blots were developed using a commercial enhanced chemiluminescence system, and densitometric analysis was performed using the Image Analyzer system (Syngene, UK).
Image analyzer system
Manufactured by Syngene
Sourced in United Kingdom
The image analyzer system is a specialized laboratory equipment designed for the analysis and processing of digital images. It provides advanced tools for image capture, enhancement, measurement, and quantification. The core function of this system is to enable researchers and analysts to extract meaningful data and insights from visual information.
Automatically generated - may contain errors
Lab products found in correlation
Igf 1, by Abcam (1 mentions)
Tissue tearor, by Biospec (1 mentions)
Tgf β1, by Abcam (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
Cleaved parp1, by Abcam (1 mentions)
Sc 7273, by Santa Cruz Biotechnology (1 mentions)
Ccaat enhancer binding protein alpha c ebpα, by Cell Signaling Technology (1 mentions)
Goat anti rabbit immunoglobulin g igg, by Cell Signaling Technology (1 mentions)
E cadherin, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
3 protocols using image analyzer system
1
Quantifying Hair Growth Regulators
2
Retinal Protein Expression Analysis
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Protein extraction and the Western blotting were performed as previously stated [32 (link)]. Retinal tissues were lysed in RIPA buffer, and the total protein value was analyzed using a standard bicinchoninic acid assay (Pierce). Sample buffer was supplemented to retinal tissue, including 30 μg of total protein. The protein was isolated utilizing 10% SDS-PAGE and fixed onto a nitrocellulose membrane. The membranes were rinsed and treated with 5% skim milk in Tris-buffered saline/Tween 20 (TBST) buffer for 1 h at room temperature. Then, the membranes were put with antibodies with regard to PARP-1 (Cell Signaling, Danvers, MA, USA, #9542; 1:1000), cleaved PARP-1 (Abcam, Cambridge, UK, #ab32064; 1:1000), and actin (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-47778; 1:200) overnight at 4 °C. The membranes were immersed in the TBST buffer containing 5% skim milk and a horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG as the secondary antibody for 1 h. Proteins were detected by ECL Western blotting substrate (Thermo Scientific, Waltham, MA, USA), and immunoblot bands were checked by an image analyzer system (Syngene, Cambridge, UK). Quantification was performed utilizing ImageJ software (NIH, Bethesda, MD, USA).
3
Protein Expression Analysis in Cells
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Cells were washed twice with phosphate buffered saline (PBS) at 4°C and lysed in RIPA buffer. Protein levels in the cell lysates were measured using a standard Bradford assay. Total protein amount (10 μg) from each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with BSA in Tris-buffered saline, 0.1% Tween® 20 Detergent (TBST) for 2 hours at RT, the membranes were incubated overnight at 4°C with specific primary antibodies: PPAR-γ (1:1,000; sc-7273; Santa Cruz Biotechnology, Dallas, TX, USA), CCAAT/enhancer binding protein alpha (C/EBPα; 1:500; D56F10, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1,000; ab40772, Abcam, Cambridge, UK), N-cadherin (1:1,000; 13116S, Cell Signaling Technology), Vimentin (1:1,000; 5741S, Cell Signaling Technology), Snail (1:1,000; Novus, NBP2-27293, Littleton, CO, USA), p-P38 (1:1,000; 4511, Novus), total-P38 (1:1,000; 9212, Novus), MMP-2 (1:1,000; GTX-104577, Genetex, Irvine, CA, USA), and β-actin (1:1,000; ab8227, Abcam). The membranes were then washed and incubated with the secondary antibodies goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG (1:2,000; Cell Signaling Technology) for 2 hours with BSA in TBST. The bands were detected using an image analyzer system (Syngene, Cambridge, UK).
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