Total protein was extracted from the cells using RIPA lysis buffer (Cell Signaling Technology, Inc.) and quantified using a BCA kit (Beyotime Institute of Biotechnology). Total protein (30 µg/lane) was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% milk for 1 h at 37˚C and then incubated in the primary antibodies, against RAB34 (1:1,000; cat. no. PA5-99697; Thermo Fisher Scientific, Inc.), Bcl-2 (1:1,000; cat. no. ab32124; Abcam), Bax (1:1,000; cat. no. 182733; Abcam), CDK4 (1:1,000; cat. no. ab108357; Abcam), CDK8 (1:1,000; cat. no. ab229192; Abcam), cyclin D1 (1:1,000; cat. no. ab16663; Abcam), E2F1 (1:1,000; cat. no. ab288369; Abcam) or GAPDH (1:1,000; cat. no. ab9485; Abcam), overnight at 4˚C. On the next day, membranes were incubated with the HRP-conjugated secondary antibody (goat anti-rabbit; 1:5,000; cat. no. ab6721; Abcam) for 2 h at 37˚C. Finally, all the membranes were visualized using a BeyoECL Plus kit (Beyotime Institute of Biotechnology) on an Odyssey Infrared imaging system (Bio-Rad Laboratories, Inc.) and semi-quantified using ImageJ software (version 1.42; National Institutes of Health).
Odyssey infrared imaging system
Manufactured by Bio-Rad
Sourced in United States
The Odyssey infrared imaging system is a lab equipment product designed for fluorescence imaging and quantitative Western blotting. The system utilizes near-infrared fluorescent dyes to detect and analyze protein targets. It is capable of high-resolution imaging and precise quantification of protein levels.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Bca kit, by Beyotime (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Bcl 2, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti tfap4, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti c myc, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg, by Abcam (1 mentions)
Anti p21, by Abcam (1 mentions)
Anti foxk1, by Abcam (1 mentions)
5 protocols using odyssey infrared imaging system
1
Western Blot Analysis of Cell Signaling
2
Western Blot Protein Quantification
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Following the published method [17 (link)], 30 μg of protein extract was prepared and processed for Western blotting analysis. Briefly, the protein on the membrane reacted with the primary antibody at room temperature for 1 h, then washed three times, and then reacted with the secondary antibody for another 1 h. As directed by the Odyssey infrared imaging system (BIO-RAD, Hercules, CA, USA), the blot was washed three times followed by development and exposure.
3
Western Blot Analysis of LPS-Stimulated Spleen Tissue
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Spleen tissue was incubated with LPS for 24 h, then protein extracts were prepared and processed for western blotting analysis according to a previously published method (Hagiwara et al., 2008 (link)). Briefly, proteins on membranes were reacted with primary antibodies at room temperature for 1 h, washed thrice, and then reacted for 1 h with the secondary antibodies. The blots were then washed thrice and developed and exposed according to the instructions of the Odyssey infrared imaging system (BIO-RAD, Hercules, CA, United States).
4
Quantifying Intestinal Organoid Proteins
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Intestinal organoid samples were collected, and the protein levels were determined as described previously.25 (link) Briefly, samples were lysed with RIPA lysis buffer (strong, Beyotime Biotechnology, Shanghai, China), and boiled at 95 °C for 10 min. Then, lysates were subjected to SDS-PAGE, followed by the transferring of proteins to a polyvinylidene difluoride (PVDF) membrane (Immobilon-FL). Then, the membrane was blocked with 5% BAS solution diluted with TBS containing 0.05% Tween 20 (TBST) at room temperature for 1 h, followed by incubation with the corresponding primary antibodies including Lgr5 (Rabbit, Miltenyi Biotec, 1 : 1000), EphB3 (Mouse, SANTA CRUZ, 1 : 1000) and GAPDH (Rabbit, Cell Signaling Technology, 1 : 1000) overnight at 4 °C. Afterwards, the membranes were washed with TBST three times after being incubated with the corresponding secondary antibodies, followed by measurement of the immunoreactive bands using an Odyssey infrared imaging system (Bio-Rad).
5
Western Blot Analysis of Protein Expression
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Cells and tissues were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology). After quantification with a BCA kit, total proteins (10 µg/lane) were fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins 10% were transferred onto a PVDF membrane (EMD Millipore), blocked with 5% skim milk for 1 h at 37˚C, and the membrane was incubated with the following primary rabbit antibodies at 4˚C overnight: Anti-TFAP4 (1:1,000; product code ab66626), anti-FOXK1 (1:1,000; product code ab85999), anti-c-Myc (1:1,000; product code ab32072), anti-p-21 (1:1,000; product code ab227443), anti-E-cadherin (1:500; product code ab15148), anti-N-cadherin (1:1,000; product code ab76057) and anti-GAPDH (1:500; product code ab8245; all from Abcam). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:2,000; product code ab6721; Abcam) for 1 h at room temperature. Using the ECL chemiluminescent kit (Genview Corporation), protein bands were visualized. The expression levels of proteins were detected and analyzed by an Odyssey Infrared imaging system (Bio-Rad Laboratories, Inc.) and ImageJ software (version 1.42; National Institutes of Health).
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