Western blotting was performed as described before (Xue et al., 2017 (link)). Each lane was loaded 20–40 μg protein, and the PVDF membrane was blocked with 5% skim milk in 0.1% TBST for 1 h. After washing the membrane for three times, the membrane was incubated with primary antibody at 4°C overnight. HRP-conjugated secondary antibody and ECL kit were used to detect primary antibody with Bio-Rad Exposure System. The gray value of each lane was calculated with ImageJ.
Exposure system
Manufactured by Bio-Rad
Sourced in United States
The Exposure system is a laboratory equipment designed for the exposure and visualization of blotted samples, such as those used in Western blotting or other analytical techniques. It provides a controlled environment for the capture and documentation of chemiluminescent or fluorescent signals from labeled proteins or nucleic acids on membranes.
Automatically generated - may contain errors
Lab products found in correlation
Ecl chemiluminescent substrate, by Bio-Rad (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Ab102536, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Goat anti rabbit igg, by Proteintech (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Affinity Biosciences (1 mentions)
Gapdh, by Huabio (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab48050, by Abcam (1 mentions)
6 protocols using exposure system
1
Western Blotting Protocol for Protein Analysis
2
Western Blot Analysis of Chondrocytes
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After pretreatment of chondrocytes, the cells were lysed with the lysate reagent which containing RIPA lysis buffer, phosphatase inhibitors, and protease inhibitors. Then collected the cells and lysate mixture into EP tubes for further lysing with an ultrasonic disruptor. Collected the supernatant after centrifugation and measured the concentration of protein samples with a microplate reader. Next, the protein samples were cooked at 100°C for 5 min after thoroughly mixed with the loading buffer. Total protein samples were loaded on the SDS-PAGE with 5% acrylamide in the stacking gel and 8.0–12.5% in the separation gel for electrophoresis. Then proteins were transferred to PVDF membranes. After 1 h of blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C and incubated with the corresponding proportion of secondary antibodies for 1 h at 25°C. Finally, a exposure system (Bio-Rad, Hercules, CA, USA) was employed to visualize the target protein bands with the assistant of ECL chemiluminescent substrate (Bio-Rad, Hercules, CA, USA).
3
GANT61 Alters Protein Expression in Daoy Cells
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Daoy cells were synchronized in RPMI 1640 medium with 10% FBS, followed by exposure to different concentrations of GANT61 for 24 h, while the control was not treated with any GANT61. The protein profile in the samples was examined by western blot analysis. Briefly, cells were collected and washed three times with PBS. Next, the cells were lysed in fresh radioimmunoprecipitation assay protein lysis buffer containing phenylmethylsulfonyl fluoride (ratio, 100:1) on ice. The total protein concentration was determined by the BCA method (ab102536; Abcam). Following separation by 10% SDS-PAGE, the samples were transferred to polyvinylidene difluoride films. Protein blots were visualized by Ponceau S staining. The films were subsequently blocked with 5% non-fat milk for 2 h at room temperature. Anti-Gli1 (1:500) and anti-CyclinD1 (1:1,000) protein antibodies were added and incubated overnight at 4°C. The films were then incubated with the secondary antibody (1:10,000) at room temperature for 1 h and washed three times with Tris-buffered saline/Tween 20 buffer. An enhanced chemiluminescence reagent (WBKLS0500; Merck Millipore, Billerica, MA, USA) was used to detect the protein levels, which were scanned using a Bio-Rad exposure system, and Image Lab 3.0 software used for quantification (Bio-Rad Laboratories, Inc.).
4
Chondrocytes Protein Extraction and Western Blot
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Chondrocytes with different pretreatment were lysed with RIPA buffer containing 1% phosphatase and protease inhibitors on the ice for 15 min. Then the cells and lysate mixture were further lysed with an ultrasonic disruptor. Centrifuged the mixture samples and collected supernatant as the protein samples. BCA protein assay kit was applied to measure the protein concentration with the help of a microplate reader. Denatured the protein samples which were mixed with protein loading buffer at 100°C for 5 min. Protein samples of 15 µg were separated by SDS-PAGE gels and electrotransfered to PVDF membranes (Millipore, USA). Afterwards, the membranes were blocked with 5% skim milk at room temperature for 1 h, next incubated with corresponding primary antibodies (Table 1
5
Proteasome and Apoptosis Pathway Analysis
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The proteins were extracted from lung tissue or cells and lysed by using RIPA buffer (high; cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) on ice for >30 min. Protein levels were quantified using BCA reagent. Samples were loaded 10 µl per lane and electrophoresed by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat milk for 2 h at room temperature. The membranes were incubated with primary antibodies at 4˚C overnight. The primary antibodies used were: 20S proteasome β1 (1:500; cat. no. sc-374405; Santa Cruz Biotechnology, Inc.), iNOS (1:1,000; cat. no. AF0199; Affinity Biosciences), cleaved caspase 3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), p-JNK (1:1,000; cat. no. ET1609-42; HUABIO, Inc.), total JNK Antibody (1:500; cat. no. ET1601-28; HUABIO, Inc.) and GAPDH (1:5,000; cat. no. 60004-1-lg; ProteinTech Group, Inc.). The secondary antibodies used were horseradish peroxide-conjugated goat anti-mouse IgG (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) and goat anti-rabbit IgG (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.). The bands were visualized using the Chemiluminescent Substrate kit (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.) combined with a Bio-Rad exposure system (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ 1.53e.
6
Quantification of Retinal Inflammation Markers
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Retinal hemispheres containing the injection area were isolated from RCS rats or rd1 mice at PO 2w, 4w, and 8w. Tissue lysis buffer containing 10% PMSF and 90% RIPA was added to extract protein. Proteins (30 μg per well) were separated on a 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% BSA for 1 h at 37 °C, the membranes were incubated with primary antibodies, including rabbit anti-Iba1 (Wako, 016-20001, 1:1000), rabbit anti-GFAP (Abcam, ab48050, 1:2000), mouse anti-GAPDH (Abcam, ab8245, 1:2000), and goat anti-TSPO (Aviva Systems Biology, OALA05219, 1:1000), overnight at 4 °C. The following day, the membranes were washed and incubated with goat anti-rabbit IgG secondary antibody (Invitrogen, #31460, 1:2000) or goat anti-mouse IgG secondary antibody (Invitrogen, 62-6520, 1:2000) or rabbit anti-goat IgG secondary antibody (Invitrogen, 81-1620, 1:2000) for 2 h at room temperature. Finally, the proteins on the membranes were detected using a Pierce™ ECL Western Blotting Substrate (Thermo, 32106) and scanned using a Bio-Rad exposure system (Bio-Rad). Relative protein expression levels were quantified using ImageJ software (NIH) with GAPDH as control. Uncropped images of western blotting and gel are shown in Supplementary Fig. 8 .
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