Protein samples were resolved by electrophoresis on 8, 10 or 13 % polyacrylamide gels as indicated, then electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5 % milk/Tris-buffered saline (TBS) then incubated overnight at 4 °C with primary antibodies diluted in 5 % milk/TBS (non-phospho antibodies) or 5 % BSA/TBS (phospho antibodies). Following washing, blots were incubated with HRP conjugated goat anti-mouse or donkey anti-rabbit secondary antibodies (Jackson Immuno Research Labs, West Grove, PA) diluted in 5 % milk/TBS for 1 h. Following washing, protein bands were visualized using Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA) and images were captured using the GeneGnome XRQ system and GeneTools software (Syngene, Frederick, MD).
Genegnome xrq system
Manufactured by Syngene
Sourced in United Kingdom, United States
The GeneGnome XRQ system is a compact, fully automated chemiluminescence imaging platform designed for high-performance protein and nucleic acid detection and analysis. The system combines advanced optics, sensitive CCD camera, and user-friendly software to capture, process, and analyze images of chemiluminescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Western lightning plus ecl reagent, by PerkinElmer (6 mentions)
Genetools software, by Syngene (6 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Radioimmunoprecipitation buffer, by Beyotime (2 mentions)
Bca protein assay kit, by CWBIO (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Jackson ImmunoResearch (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lamin b1, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Loading buffer, by Wuhan Servicebio Technology (1 mentions)
β tubulin, by Proteintech (1 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (1 mentions)
Bicinchoninic acid (bca), by Wuhan Servicebio Technology (1 mentions)
P ikbα, by Cell Signaling Technology (1 mentions)
P ikkα β, by Cell Signaling Technology (1 mentions)
Il 1β, by ABclonal (1 mentions)
Immobilon ecl ultra western hrp substrate, by Merck Group (1 mentions)
15 protocols using genegnome xrq system
1
Western Blot Protocol for Protein Analysis
2
Western Blot Analysis of Liver Proteins
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Proteins were extracted from the liver tissues and stored at −80 °C. For western blotting analysis, 60 µg of total protein were loaded and separated with a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1.5 h prior to being incubated overnight at 4 °C with antibodies against Bax (1:2,000, CST), Bcl-2 (1:1,000, Abcam), Nrf2 (1:500, Abcam), HO-1 (1:1,000, Abcam), NQO1 (1:2,000, Abcam), (p-Akt (1:1,000, CST), Akt (1:3,000, CST), p-GSK3β (1:500, CST), GSK3β (1:2,000, CST), β-actin (1:10,000, Abcam), and/or Lamin B1 (1:1,000, Abcam). Finally, the membranes were incubated with an HRP-conjugated goat anti-rabbit/mouse polyclonal secondary antibody (1:10,000, ASPEN) for 2 h at room temperature. Protein bands were visualized with a GeneGnome XRQ system (Syngene, UK).
3
Protein Extraction and Western Blot Analysis
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Cellular protein was extracted using RIPA Lysis Buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA and 1% NP-40, Servicebio, Wuhan, China) and protease inhibitor cocktail, and the protein concentration was detected using BCA (Servicebio, Wuhan, China). Loading buffer (Servicebio, Wuhan, China) was added before boiling for 10 min for use. For electrophoresis, 10% SDS-PAGE gel was used transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skimmed milk powder. Primary antibodies used for incubation were PKCθ (13643, Cell Signaling Technology, Danvers, MA, USA), IKKα (2682, Cell Signaling Technology, Danvers, MA, USA), p-IKKα/β (2697, Cell Signaling Technology, Danvers, MA, USA), IKBα (10268-1-AP, Proteintech, Wuhan, China), p-IKBα (2859, Cell Signaling Technology, Danvers, MA, USA), P65 (ab7970, ABCAm, Cambridge, UK), p-P65 (3033, Cell Signaling Technology, Danvers, MA, USA), IL-1β (A16288, Abclonal, Wuhan, China), and β-Tubulin (10094-1-AP, Proteintech, Wuhan, China). After washing off the primary antibody with PBST, the HRP-conjugated anti-rabbit or mouse antibody (Proteintech, Wuhan, China) was incubated for 2 h at room temperature. After adding ECL Chemiluminescent kit (NCM Biotech, Suzhou, China), the target proteins were visualized using the GeneGnome XRQ system (Syngene, Cambridge, UK).
4
Protein Expression Analysis Protocol
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Cells were harvested and lysed in radioimmunoprecipitation buffer (Beyotime, China). Quantification was performed using the BCA protein assay kit (CWBIO, China). Proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA), after blocking with 2% skim milk or bovine serum albumin (BSA). The membrane was then treated with primary antibodies at 4°C overnight and then washed using 1× Tris-Bufferd Saline and Tween 20 (TBST, CWBIO, China). In the end the membranes were incubated with secondary antibodies (EMAR, China) for 1 h at room temperature. The protein expression level was measured by Immobilon ECL Ultra Western HRP Substrate (Millipore, USA) using GeneGnome XRQ system (Syngene, USA).
5
Protein Expression Analysis in Kidney Tissue
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Proteins from kidney tissues and cultured cells were extracted with RIPA lysis buffer (Beyotime). Protease and phosphatase inhibitor cocktails were added for the extraction of phosphorylated proteins. The total protein concentration was measured using a BCA protein assay kit (Beyotime). Proteins (40–80 μg) were separated using SDS-PAGE (10%) and transferred to a PVDF membrane. The membranes were blocked with 5% BSA for 1.5 h and incubated with primary antibodies overnight at 4°C, followed by secondary antibodies (goat anti-rabbit, 1:3000; anti-mouse, 1:5000; Servicebio, Wuhan, China) for 1.5 h. The following primary antibodies were used: anti-α-SMA (1:3000, Proteintech, Wuhan, China), anti-Col1a1 (1:1000, Cell Signaling Technology), anti-collagen type III alpha 1 chain (anti-Col3a1, 1:1000, Abclonal), anti-Cpt1a (1:1000, Abclonal), anti-Acox1 (1:1000, Abclonal), anti-phosphorylation-AMP-activated protein kinase (anti-p-AMPK, 1:1000, Abclonal), and anti-glyceraldehyde-phosphate dehydrogenase (anti-GAPDH, 1:50000, Abclonal). Images were visualized using a Gene Gnome XRQ system (Syngene, Cambridge, UK).
6
Western Blot Protein Detection
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Protein samples were resolved by electrophoresis on 10% polyacrylamide gels, then electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5% milk/Tris-buffered saline (TBS) and then incubated overnight at 4 °C with primary antibodies diluted in 5% BSA/TBS. Following washing, blots were incubated with HRP conjugated goat anti-mouse IgG/IgM (heavy and light chains, but pre-absorbed to human, bovine and horse serum proteins) or goat anti-rabbit secondary antibodies (Jackson Immuno Research Labs, West Grove, PA) diluted in 5% milk/TBS for 1 h. Following washing, protein bands were visualized using Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA), and images were captured using the GeneGnome XRQ system and GeneTools software (Syngene, Frederick, MD).
7
Protein Quantification and Western Blot
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Total protein from gastric mucosal homogenates or cultured cells was extracted using a Cell Lysis Buffer (P0013J, Beyotime), and the BCA Protein Assay Kit (P0010S, Beyotime) was used for protein quantification. 40 μg of protein were added per hole and separated by 4%–20% SDS-PAGE and then transferred to 0.45 μm PVDF membranes (Millipore). Membranes were blocked in 5% skim milk at normal temperature for 1 h and incubated with the primary antibodies (CLEC4E, 1:1000) overnight at 4 °C followed by the second antibody incubation. The β-actin was used as an internal control. The protein bands were photographed using the GeneGnome XRQ system (Syngene, Cambridge, UK) and analyzed using ImageJ software (NIH, MD, USA).
8
Western Blot Analysis of CRC Proteins
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Whole-cell lysates of CRC cell lines and xenograft tissues were prepared in Complete Lysis-M buffer (Roche, Germany). Protein quantification was performed using the Pierce™ BCA protein assay (Thermo Fisher Scientific, UK). 30 µg of total protein per lane was loaded on acrylamide gel. Following the SDS-PAGE electrophoresis, proteins were transferred to a nitrocellulose membrane (Geneflow, UK), blocked for 1 h in 5% milk and incubated overnight with primary antibodies at 4 °C. Membranes were then washed with PBS-0,01%Tween (SIGMA, UK) and incubated with appropriate secondary antibodies at room temperature for 1 h. Proteins were visualized using the Enhanced Chemiluminescence Luminol (ECL) reagent (Geneflow Ltd., UK). Images were captured using the GeneGnome XRQ system (Syngene, UK) or Kodak X-Ray films. The protein band intensity was measured using ImageJ software and the relative expression levels of the proteins were normalized against actin. The protein band intensity was measured using densitometry performed with ImageJ software. Proteins presented as double bands were selected and quantified as a whole. The relative expression levels of the proteins were normalized against actin.
9
Protein Extraction and Western Blot Analysis
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After removing the supernatant, the cells were washed twice with PBS at 4°C. RIPA lysis buffer (Beyotime, China) containing 1% protease inhibitor (Sigma–Aldrich, USA) and 1% serine protease inhibitor (Sigma–Aldrich, USA) was added for 30 min to lyse cells and extract proteins from each sample. The concentrations of total proteins were detected by a BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Protein samples were mixed with 5× loading buffer (ThermoFisher, USA) at a ratio of 4:1 and boiled at 99°C for 10 min. Samples were loaded and run on SDS–PAGE gels (CWBIO, China) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 2% skim milk (BD, USA) for 1 h at room temperature and then incubated with anti-IKKα, anti-IKKβ, anti-pIKKα/β, anti-p65, anti-pp65, anti-p38, anti-pp38, anti-SAPK/JNK, anti-pSAPK/JNK, and anti-GAPDH primary antibodies (Abcam, UK) overnight at 4°C. After primary incubation, blots were washed and incubated with secondary goat anti-rabbit or goat anti-mouse HRP (Abcam, UK) for 1 hour. Membranes were washed and exposed to chemiluminescent HRP substrate (Millipore, USA). Images were obtained using the GeneGnome XRQ system (Syngene, USA) and analyzed using ImageJ software.
10
Spinal Cord Protein Extraction and Western Blot
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