Western blots were performed as previously described (32 (link)) using NuPAGE (Invitrogen) gels and transfer to polyvinylidene difluoride (PVDF). Membranes were blocked in 5% nonfat dried milk in TBST (Tris-buffered saline [pH 7.4] with 0.05% Tween 20) and then incubated with primary antibodies as follows: RB1 (Calbiochem/EMD), UBR4 (gift of Yoshihiro Nakatani, Dana-Farber Cancer Institute [67 (link)]), CUL3 (Bethyl), actin (Millipore), V5 (Invitrogen), p53 (Santa Cruz Biotechnology), or PTPN14 (Sigma-Aldrich, R&D Systems, or Cell Signaling Technology). Membranes were washed in TBST and incubated with horseradish peroxidase (HRP)-coupled anti-mouse or anti-rabbit antibodies or an Alexa 680-coupled anti-mouse antibody and detected using Western Lightning chemiluminescent substrate or a LI-COR infrared imaging system. HA-tagged proteins were detected using an HA antibody conjugated to HRP (Roche) and visualized on film. For anti-HA immunoprecipitations, HA-tagged proteins were immunoprecipitated and processed for Western blotting as previously described (32 (link)).
Infrared imaging system
Manufactured by LI COR
Sourced in United States, China
The Infrared Imaging System is a laboratory equipment designed to capture and analyze infrared images. It uses specialized sensors to detect and record infrared radiation, providing detailed information about the thermal characteristics of the subject under observation.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Nitrocellulose membrane, by Merck Group (7 mentions)
Odyssey blocking buffer, by LI COR (5 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
β actin, by Abcam (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Irdye 800cw, by LI COR (3 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (3 mentions)
Exoab cd63a 1, by System Biosciences (3 mentions)
Odyssey 3, by LI COR (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Odyssey software, by LI COR (2 mentions)
Odyssey blocking buffer tbs, by LI COR (2 mentions)
Fluorescent conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Sds polyacrylamide gel, by Bio-Rad (2 mentions)
88 protocols using infrared imaging system
1
Western Blot Protocol for Protein Detection
2
Western Blot Analysis of STAT1 in LAC Cells
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All proteins were obtained from LAC cells and patients. A549 cells and patient tissues were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). The proteins were then quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). The proteins (60–80 µg) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Following blocking with 5% non-fat milk for 2 h at room temperature, the blots were probed with primary antibodies against STAT1 (catalog no. ab30645; 1:500; Abcam, Cambridge, MA, USA) and β-actin (catalog no. 4970; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Following threes washes with PBS and Tween-20 for 15 min, the membranes were incubated with rabbit (catalog no. 926-32211-00; 1:10,000) or mouse (catalog no. 926-32211-01; 1:10,000) secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) at room temperature in the dark for 1 h. The blots were then visualized using an Infrared Imaging System (LI-COR Biosciences) and the band density was quantified using Odyssey 3.0 software (LI-COR Biosciences) (22 (link)). Using β-actin as an internal control, the blots were subjected to densitometry.
3
Prostate Acid Phosphatase Protein Expression
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The testicular tissue was lysed with a RIPA buffer with protease inhibitors. The total soluble protein was quantified by the BCA protein assay. The total protein (30 μg) was loaded onto a 10% SDS-PAGE gel, separated by electrophoresis, and transferred onto a polyvinylidene difluoride membrane. The blots were blocked with 5% skim milk and incubated with a primary antibody overnight at 4 °C, followed by incubation with a secondary antibody for 1 h at room temperature, and measured with an Infrared Imaging System (LI-COR, Lincoln, NE, USA). The protein expression was normalized by the detection of β-actin (Abcam, Cambridge, UK). The protein from the testicular tissue was isolated to measure the expression level of PAP, and was probed using the primary monoclonal antibody (Santa Cruz, CA, USA).
4
Protein Expression Analysis in Cells
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Cells were lysed in RIPA buffer (Beyotime, Nantong, China) supplemented with phenylmethylsulfonyl fluoride (Beyotime, Nantong, China). Proteins were then separated by 15% SDS-PAGE gel and transferred onto the PVDF membrane (Millipore, Billerica, MA, USA). β-actin (Abcam, Cambridge, MA, USA), JAK2 (Abcam, Cambridge, MA, USA), STAT1 (Abcam, Cambridge, MA, USA), IFN-γ (Abcam, Cambridge, MA, USA), IL-4 (Abcam, Cambridge, MA, USA), S100A4 (Abcam, Cambridge, MA, USA), p-JAK2 (Abcam, Cambridge, MA, USA) and p-STAT1 (Abcam, Cambridge, MA, USA) primary antibodies and fluorescent dye-labeled secondary antibody were used. The density of protein bands was measured by an infrared imaging system (LI-COR, Lincoln, NE, USA).
5
Monitoring RING2(Rcat)-Ubiquitin Ester Formation
6
Immunoblotting and Immunoprecipitation Analysis
7
Quantification of AANAT Protein Levels
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Proteins from BON-1 cells were extracted with RIPA extraction buffer mixed with a protease inhibitor cocktail. Equal amounts of protein (30 µg) were electrophoresed in 12% sodium dodecyl sulfate polyacrylamide gels and transferred onto nitrocellulose membranes under a constant current of 250 mA for 90 min. The membranes were incubated with rabbit anti-AANAT antibody (1:1000; catalog No. ab3505, Abcam) overnight at 4 °C. β-Actin was chosen as the internal reference protein. The primary antibodies were detected using anti-rabbit or mouse secondary antibodies and were visualized with an infrared imaging system (LI-COR Biosciences, Lincoln). The band intensity relative to that of β-actin was calculated, and the results were expressed as fold change from control values.
8
Quantification of Insulin Receptor Protein
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Cells were lysed with RIPA buffer with protease inhibitors. Total soluble protein was quantified by the BCA protein assay. Total protein (30 μg) was loaded onto a 10% SDS-PAGE gel, separated by electrophoresis, and transferred onto a polyvinylidene difluoride membrane. Blots were blocked with 5% skim milk and incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody for 1 h at room temperature, and measured with an Infrared Imaging System (LI-COR, Lincoln, NE). Protein expression was normalized by detection of β-actin (Abcam, Cambridge, UK). Protein from cells treated with TNF-α/DMEM-F12 and inducted to day 8 was isolated to measure the expression level of IR.
9
Brain Protein Extraction and Western Blot Analysis
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Total protein from the brain of each mouse was extracted with RIPA buffer and quantified using a Protein Reagent Assay BCA Kit (Thermo, Waltham, MA, USA). Thirty micrograms of protein from each sample was loaded and electrophoresed using 15% SDS-PAGE gels and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After the membranes were blocked with 3% bovine serum albumin (BSA) at room temperature for 60 min, they were incubated with primary antibodies overnight at 4 °C. Then, the blots were incubated with secondary antibody for 2 h at room temperature (see Additional file 3 : Table S2 for the antibodies used). The blots were visualized using an Infrared Imaging System (Odyssey, LI-COR, NE Lincoln, USA).
10
Immunoblotting of Cell Lysates
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