Cultured CRC cells treated without or with selumetinib (500 nM) at 37°C for 24 h were collected and lysed with 1X RIPA Lysis Buffer (Sigma-Aldrich; Merck KGaA) containing 1X Protease Inhibitor Cocktail (Sigma-Aldrich; Merck KGaA). The concentration of total protein was measured with a BCA protein assay kit (Beyotime Institute of Biotechnology). Total protein (30 µg/lane) was separated using 6–12.5% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and blocked with 5% skimmed milk at room temperature for 1 h. Next, membranes were incubated with primary antibodies against CRMP5 (Abcam; cat. no. ab36203; 1:2,000), Ki67 (Abcam; cat. no. ab15580; 1:1,000), Caspase-3 (CST; cat. no. 96659665; 1:1,000), E-cadherin (CST; cat. no. 3195; 1:1,000), MMP-2 (CST; cat. no. 4022; 1:1,000), MMP-9 (CST; cat. no. 13667; 1:1,000) and vimentin (CST; cat. no. 5741; 1:1,000) overnight at 4°C, followed by incubation with fluorescence-conjugated goat ant-mouse IgG H&L (Abcam; cat. no. ab216772) or goat an-rabbit IgG H&L (Abcam; cat. no. ab216773) secondary antibodies (dilution, 1:1,000) at room temperature for 1 h. Finally, bands were detected using a two-color infrared laser imaging system (Odyssey; LI-COR Biosciences).
Two color infrared laser imaging system
Manufactured by LI COR
Sourced in United States, Germany
The Two-color infrared laser imaging system is a laboratory instrument designed for visualizing and analyzing biological samples. The system utilizes two infrared lasers to simultaneously capture images of labeled targets within a sample. The core function of the system is to provide high-resolution, multi-channel imaging capabilities for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Pdvf membrane, by Bio-Rad (1 mentions)
Wet transfer method, by Bio-Rad (1 mentions)
Sds page, by Beyotime (1 mentions)
4 protocols using two color infrared laser imaging system
1
Selumetinib Modulates CRC Cell Signaling
2
Western Blot Analysis of HAX-1 Protein
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Protein samples were isolated with lysis buffer (RIPA) containing protease inhibitors. After quantification with a BCA kit (Beyotime, China), total protein (25 μg) was separated by 8–15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skimmed milk for 60 min, the membranes were incubated with primary antibodies anti-HAX-1 (ab137613, 1 : 500) and anti-GAPDH (#5174, CST, 1 : 1000) overnight, followed by incubation with fluorescence-conjugated secondary antibodies (1 : 1,000) for 30 min. Bands were detected by using a two-color infrared laser imaging system (Odyssey; Li-Cor, Lincoln, NE, USA) [20 (link)].
3
Intestinal Protein Expression Analysis
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Tissue proteins were extracted after homogenizing the small intestinal tissues. The denatured proteins were separated using 12% SDS-PAGE (MultiSciences Biotech, China). Subsequently, the proteins on the gel were transferred to the PDVF membranes (Bio-Rad, USA). Following the blocking step with 5% skimmed milk powder for 1 h, the membranes were incubated with primary antibody (1 500 ZenBio, China) overnight at 4°C. The membranes were then incubated with secondary antibody Goat Anti-Mouse IgG (ZenBio, China) for 1 h for protein labeling. The protein bands were developed using a chromogenic agent (Beyotime, China) and photographed in a two-color infrared laser imaging system (LI-COR, Germany). Each sample was analyzed in triplicate.
4
SDS-PAGE and Western Blot Analysis
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The denatured proteins were separated with 12% SDS‐PAGE (Beyotime, China). The electrophoretic conditions were 100V for 20 min and 120 V for 60 min. The protein was transferred to PDVF membrane by wet transfer method (BioRad). The membrane was incubated with the first antibody, and then secondary antibody (ZenBio) was used for protein labeling. The protein bands were developed using the color‐substrate solution (Beyotime) and photographed in a two‐color infrared laser imaging system (Li‐cor).
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