Total protein was extracted from cells with 1% RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China); protein quantitation was carried out by the BCA method. Equal amounts of protein (30 µg) were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, the samples were incubated with relevant antibodies, including anti-HMGB1 (cat. no. ab79823, 1:1,000; Abcam, Cambridge, MA, USA), anti-MMP2 (cat. no. 40994, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-MMP9 (cat. no. 13667, 1:1,000; Cell Signaling Technology) anti-cyclin D1 (1 µg/ml; cat. no. AF4196; R&D Systems, Minneapolis, MN, USA) and anti-GAPDH (cat. no. GTX100118, 1:5,000; GeneTex, Inc., Irvine, CA, USA), overnight at 4°C. This was followed by incubation with HRP-labeled goat anti-rabbit or anti-rat IgG (H+L) (1:5,000; Beyotime Institute of Biotechnology) for 60 min at room temperature. Immunodetection was performed by enhanced chemiluminescence (ECL Plus kit; Beyotime Institute of Biotechnology) and visualized on a G:Box Chemi imaging system (Syngene, Cambridge, UK).
G box chemi imaging system
Manufactured by Syngene
Sourced in United Kingdom, India
The G:BOX Chemi imaging system is a versatile laboratory equipment for capturing and analyzing images of gels, blots, and other samples. It provides a platform for researchers to visualize and document their experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop, by Roche (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by R&D Systems (1 mentions)
Anti mmp9, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl plus kit, by Beyotime (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Ripa lysis buffer, by CoWin Biotech (1 mentions)
Secondary antibodies conjugated to horseradish peroxidase, by GE Healthcare (1 mentions)
Anti gst, by GE Healthcare (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Enhanced chemiluminescence ecl kit, by GE Healthcare (1 mentions)
Anti mdm2 ab 1, by Merck Group (1 mentions)
Anti myc 9e10, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti gfp, by Roche (1 mentions)
Tween 20, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Biorupter sonicator, by Diagenode (1 mentions)
9 protocols using g box chemi imaging system
1
Protein Expression Analysis in Cells
2
Evaluating AKT Activation in iPSC-MSC Coculture
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The Lin− cells cocultured with iPSC‐MSCs were collected and lysed in RIPA lysis buffer (CoWin Biosciences, Beijing, China) supplemented with phosphatase inhibitor PhosSTOP (Roche, Basel, Switzerland). The lysates were subjected to SDS‐PAGE for detecting AKT (Akt Antibody #9272), phospho‐AKT (Phospho‐Akt (Ser473) (D9E) XP Rabbit mAb #4060, Cell Signaling Technology, Danvers, Massachusetts). β‐Actin (IGX3831H, Abcam, Cambridge, UK) was detected as the internal control. Immunoreactive bands were captured on the G:BOX Chemi Imaging System (Syngene, India) and quantified with ImageJ software.
3
Western Blot Analysis of Protein Interactions
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Total cell lysates and immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted by a semi-dry transfer in an electric field onto polyvinylidene difluoride membranes (PVDF; Millipore). After transfer, the membranes were blocked in 5% low fat dry milk in TBST (10 mM TRIS-HCl, 100 nM NaCl and 0.05% Tween 20; pH 7.4) for 1 h at room temperature and incubated overnight at 4°C with a primary antibody in 1% low fat dry milk in TBST. The following primary antibodies were used: anti-TFII-I (V-18; Santa Cruz Biotechnology), anti-GST (GE Healthcare), anti-Mdm2 (Ab-1; Merck-Millipore), anti-Tubulin alpha (Exbio), anti-PCNA, (PC-10, kindly provided by Borivoj Vojtesek, Masaryk Memorial Cancer Institute, Brno, Czech Republic), anti-β-galactosidase (BG-02; Santa Cruz Biotechnology), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Flag (M2; Sigma-Aldrich) and anti-GFP (Roche), using dilutions recommended by the manufacturers. After three washes in 1% milk in TBS, membranes were incubated one hour at room temperature with secondary antibodies conjugated to horseradish peroxidase (GE Healthcare). Enhanced chemiluminescence kit (ECL; GE Healthcare) and blue light sensitive medical X-ray film (Agfa) or G:BOX Chemi imaging system (Syngene) were used to visualize proteins of interest.
4
Protein Expression Analysis by Western Blot
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Cells were collected in sample buffer, supplemented with β-mercaptoethanol, after a rapid wash in PBS (Phosphate-Buffered Saline – Sigma-Aldrich), and then sonicated. Samples were separated by either 12 or 10% SDS-PAGE [Acrylamide/Bis-acrylamide solution 37.5:1 (Euro-Clone)], transferred to nitrocellulose membranes and blocked in 5% skimmed milk in Tris-Buffered Saline-Tween-20 (TBS-T): 20 mM Tris HCl pH 7.4, 150 mM NaCl, supplemented with 0,2% Tween-20 (Sigma-Aldrich). Blots were incubated with primary antibodies in blocking solution overnight at 4 °C, then washed in TBS-T and incubated with horseradish-conjugated secondary antibodies in blocking solution. After extensive washing, blots were developed with chemiluminescence-based detection system (StoS PSD, Genespin) coupled with G:BOX Chemi Imaging System (Syngene). Densitometric expression analyses of protein expression were performed using FiJi Software. Protein expression levels were normalised using GAPDH as internal standard. Phosphorylation was evaluated as the ratio between phosphoproteins and their total form.
5
Western Blot Analysis of Naive B Cells
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All antibodies used for western blotting are listed in Supplementary Table 3 . 0.8–1 × 106 primary naive B cells were recovered for protein extraction, which was performed using RIPA lysis buffer (Pierce) followed by cycles of sonication performed using the Biorupter Sonicator (Diagenode). Protein concentration was analysed using the BCA Protein Assay kit (cat no. 23225 from Pierce) and read by the Model 680 Microplate reader (Bio-Rad). Proteins were separated by the NuPAGE SDS-PAGE Gel system (Thermo Scientific) and transferred onto Immobilon-P PVDF membranes (Sigma-Aldrich) according to standard procedure. Detections were performed with HRP-conjugated secondary Ab (Bio-Rad) and enhanced chemiluminescent (ECL Plus) reagent (Amersham) using the G:BOX Chemi imaging system (Syngene). GAPDH or β-ACTIN on the same membrane served as loading control. Uncropped original scans of immunoblots are provided in Supplementary Fig. 9 .
6
Expression and Detection of TaPHB-1 Protein
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The recombinant pGBKT7-TaPHB-1 was transformed into a Y2HGold yeast strain following the Yeastmaker yeast transformation system 2 (TaKaRa). The transformants were screened on an SD/Trp agar plate. A colony from the SD/Trp was inoculated in 5 mL of SD/–Trp broth and cultured overnight at 30°C and 250 rpm. The overnight culture was added to 50 mL of YPDA and cultured at 30°C until the optical density at 600 nm (OD600) reached 0.4 to 0.6. The protein extract was prepared by lysing the yeast cells in a cracking buffer, as described previously (44 (link)). The protein lysate was resolved on 12% SDS-polyacrylamide gel and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane. After being blocked in blocking solution (3% skimmed milk powder in Tris-buffered saline with Tween 20 [TBST]), the membrane was incubated with myc antibody (Santa Cruz Biotechnology). The expression of myc-tagged TaPHB-1 was detected using horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Pierce) with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) in a G:BOX Chemi imaging system (Syngene).
7
CDKL5 Protein Expression Analysis
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Primary hippocampal neurons were lysed in 3X Laemmli buffer, and samples were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes and blocked in 5% non-fat milk in TBS-T (20 mM of Tris-HCl pH 7.4, 150 mM of NaCl, 0.2% Tween-20). Blots were incubated with primary antibodies overnight at 4 °C, washed in TBS-T and incubated with appropriate secondary antibodies for 1 h at room temperature. Blots were developed with protein detection system-ECL (Genespin) coupled to G:BOX Chemi Imaging System (Syngene). Densitometric expression analyses were performed using ImageJ software.
CDKL5 was immunoprecipitated from 400 μg of HEK293T cells or 1 mg of a mouse brain extract (PND20-30) lysed in lysis buffer [mM: 50 Tris-HCl pH 7.4, 150 NaCl, 1 EDTA, 1 EGTA, 1% Triton X-100, 1X protease inhibitor cocktail (PIC, Sigma-Aldrich, Sant Louis, MO 63103, USA) and 1X PhosSTOP (Roche)] and incubated overnight at 4 °C with 1 µg of anti-CDKL5 or unrelated IgGs as control. The immunocomplexes were precipitated with protein-G agarose (Life Technologies), washed several times with lysis buffer and analysed by SDS-PAGE and western blotting.
CDKL5 was immunoprecipitated from 400 μg of HEK293T cells or 1 mg of a mouse brain extract (PND20-30) lysed in lysis buffer [mM: 50 Tris-HCl pH 7.4, 150 NaCl, 1 EDTA, 1 EGTA, 1% Triton X-100, 1X protease inhibitor cocktail (PIC, Sigma-Aldrich, Sant Louis, MO 63103, USA) and 1X PhosSTOP (Roche)] and incubated overnight at 4 °C with 1 µg of anti-CDKL5 or unrelated IgGs as control. The immunocomplexes were precipitated with protein-G agarose (Life Technologies), washed several times with lysis buffer and analysed by SDS-PAGE and western blotting.
8
Western Blotting Protein Analysis
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Cells were lysed using RIPA buffer supplemented with complete, Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland, Cat. #11836153001) for 30 min on ice. Cell debris was removed by centrifugation 12,000 rpm for 20 min at 4 °C. Protein concentration were determined using Pierce BCA Protein Assay Kit (ThermoFisher, Cat. #23225), according to manufacturer’s protocol. Proteins were reduced and denatured in 4× Laemmli Sample Buffer (Bio-Rad, Cat. #1610747) supplemented with 2-mercaptoethanol (Bio-Rad Cat. #1610710XTU) at 100 °C for 10 min. A total of 25 μg of protein was resolved on a 4–15% Mini-PROTEAN TGX gel (Bio-Rad Cat. #4561083) and transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer System and Trans-Blot Turbo Transfer Packs (Bio-Rad, Cat. #170-4158). Protein expression was determined using Pierce ECL Western Blotting Substrate (ThermoFisher, Cat. #32109) and the Syngene G:BOX Chemi imaging system and GeneTools analysis software after overnight incubation with primary antibodies at 4 °C and 1 h incubation at room temperature with secondary antibody. All westerns presented in a single figure panel are from the same experiment and full uncropped images can be seen in Supplementary Figure S2 .
9
Arabidopsis Protein Expression Analysis
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Western blots were prepared from lysates from Arabidopsis rosettes incubated with or without 3',5'-cAMP (as in the TPP experiment). Each lysate was divided into four parts for different temperature treatments (50, 52, 55, 60 ºC) . The protein samples were separated by SDS-PAGE and blotted onto membranes. Immunoblot detection was performed with specific antibodies using the enhanced chemiluminescence system (GE Healthcare). Signals were detected with the G:box Chemi Imaging System (Syngene). Antibodies used for the western blots were obtained from commercial suppliers: anti-actin monoclonal antibody (Sigma-Aldrich A0480-200µL) and antimouse antibody (Agrisera AS111772). Western blot quantification was performed with the opensource Fiji software.
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