Lung tissue lysate was prepared in the 20 mM Tris–HCl (pH 7.5) buffer containing 2 mM sucrose, 2 mM EDTA, 0.5 mM EGTA, 2 mM MgCl2, 2 mM PMSF, 1 mM DTT, 0.03 mM Na3VO4 and protease inhibitor cocktails (Sigma Co., USA). Protein concentration was quantified by Bradford's method. Tissue lysate equivalent to 50 μg of protein was resolved on to 8–12.5% Tris–glycine gel and transferred onto an Immobilion-P-membrane (PVDF) (Millipore, USA) for 1 h at 90 V. Subsequently, membranes were blocked with 3% BSA in TBST (100 mM Tris–HCl, 150 mM NaCl and 0.1% Tween-20) for 1 h and probed with the primary antibodies for PCNA, cyclin D1, NF-κBp50, IL-6, STAT3, pSTAT3, pAkt, PARP, caspase-3, caspase-9, COX-2 and Akt at a dilution 1:3000. Further incubation was done with appropriate secondary antibodies conjugated to horseradish peroxidase (Bangalore Genei, India) at a dilution 1:10,000. The immunocomplex, thus formed, was visualized with the help of Super Signal West Femto Max Substrate in Versa Doc Gel Imaging System (Bio-Rad, Hercules, CA) and quantified using Gene Tool Syngene software. Each membrane was stripped and re-probed with β-actin as a loading control.
Versadoc gel imaging system
Manufactured by Bio-Rad
Sourced in United States
The VersaDoc gel imaging system is a versatile instrument designed for capturing and analyzing images of gel-based samples. It utilizes a high-resolution camera and advanced optics to provide clear and detailed images of various types of gels, including agarose, polyacrylamide, and other common gel-based samples. The VersaDoc system is capable of detecting a wide range of signals, including chemiluminescence, fluorescence, and colorimetric signals, making it a versatile tool for various applications in the laboratory.
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Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse igg antibody, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemi luminescence (ecl), by PerkinElmer (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Proteases inhibitor cocktail, by Roche (1 mentions)
Supersignal west pico sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase, by Dianova (1 mentions)
Femto sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Mouse anti gfp antibody, by Santa Cruz Biotechnology (1 mentions)
Pierce ecl western substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Talon affinity resin, by Takara Bio (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Mouse anti rev antibody, by Santa Cruz Biotechnology (1 mentions)
14 protocols using versadoc gel imaging system
1
Lung Tissue Lysate Protein Analysis
2
Western Blot Analysis of Alpha-Synuclein
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For Western blot analysis, cortex and olfactory bulb of mouse brains were homogenized in RIPA Buffer (50mM Tris/HCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) NP-40, 0.1% (w/v) SDS) in the presence of a proteases inhibitor cocktail (Roche) with a douncer homogenizer Potter at 4°C. Protein levels in the tissue homogenates were quantified via BCA protein assay (Thermo Scientific). 80 μg of total proteins were mixed with the same volume of SDS sample buffer (0.125 M Tris/HCl pH 6.8, 4% SDS, 20% glycerol), separated on 15% SDS-PAGE, and blotted onto nitrocellulose membranes (Millipore). The blots were probed with a mouse anti-alpha-synuclein-1 antibody (1:2000, BD Transduction Laboratories) and a rabbit anti GAPDH antibody (1:2000, FL335, Santa Cruz Technology), followed by the incubation with secondary goat anti mouse and goat anti rabbit antibodies coupled to horseradish peroxidase (1:10000 Dianova, Hamburg, Germany). For the detection of proteins, membranes were incubated with the SuperSignal West Pico Sensitivity Substrate (Thermo Scientific) and visualized by VersaDoc gel imaging system (BioRad).
3
Western Blot of Recombinant Alpha-Synuclein
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For Western blot (WB) of recombinant aSyn, 0.5 μg aSyn was mixed with one volume of SDS sample buffer (0.125 M Tris/HCl pH 6.8, 4% SDS, 20% glycerol), separated on 15% SDS-PAGE, and blotted onto nitrocellulose membranes (Millipore, Darmstadt, Germany). The blots were probed with a mouse anti-aSyn primary antibody (Syn-1, 1:2000, BD Transduction Laboratories, San Diego, CA, USA) or a rabbit anti-aSyn antibody (SNCA antibody, 1:2000, Proteintech Europe, Manchester, UK). While Syn-1 was generated using aSyn fragment amino acids 15-123 as antigen, SNCA antibody was raised against full-length aSyn. The nitrocellulose membranes were subsequently probed with secondary goat-anti-mouse antibody or goat anti-rabbit antibody coupled to horseradish peroxidase (1:10000 Dianova, Hamburg, Germany). For the detection of proteins, membranes were incubated with the SuperSignal West Pico or Femto Sensitivity Substrate™ (Thermo Scientific Rockford, lL, USA). Immunoblots were visualized by VersaDoc gel imaging system (BioRad, Munich, Germany).
4
Cartilage Marker Gene Expression Analysis
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To investigate gene expression profile of chondrocyte primary cultures, mRNAs of specific cartilage markers were analysed using reverse transcription polymerase chain reaction (RT-PCR). Total RNA of cultured chondrocytes was first isolated using Trizol and quantified by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) at 260 and 280 nm. Reverse transcription and specific amplification were performed in a single tube using QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany, EU) according to the manufacturer's instructions. Specific oligoprimers (Life technologies, Carlsbad, CA, USA) designed on Gene Bank sequences (Table 1 ) were used and the expression of HPRT was considered as internal control. Finally, PCR products were separated by 7% polyacrylamide gel electrophoresis and visualized by silver nitrate staining. Pictures were taken using 3000 VersaDoc Gel Imaging System (Bio-Rad, Hercules, California, USA) and Quantity One software (Bio-Rad). Finally, band intensities were quantitated by densitometry, using Image J software.
5
HIV-1 LTR Pull-Down Assay in Astrocytoma and HEK293T Cells
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The details of pull-downs from astrocytoma 1321N1 cell lysates using biotinylated HIV-1 LTR are given as File S1 , Figure S1 and S2 . Pull-down assays were also performed using cell lysates of HEK293T cells transiently expressing hZNF-134-GFP or GFP protein. The cell lysates were incubated with the biotinylated LTR immobilized to streptavidin agarose beads. The beads were washed 5 times with 1X Saline-sodium citrate buffer before adding SDS loading dye. The samples were fractionated on 10% SDS-PAGE and transferred onto nitrocellulose membrane (Pall Life sciences, USA). The Western blot was performed using 1∶1000 dilution of mouse anti GFP-antibody (SantaCruz Biotechnology Inc., USA) followed by secondary detection with 1∶2000 dilution of HRP conjugated goat anti-mouse IgG antibody (SantaCruz Biotechnology Inc., USA). This was detected using Pierce ECL western substrate (Thermo Scientific, USA) and visualized using VersaDoc gel imaging system (BioRad, USA).
6
Expression and Purification of Recombinant HIV-1 Rev Protein
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His-Rev-setB construct was transformed into BL-21 DE3 codon plus (RIL) cells (Agilent technologies, USA) for expressing recombinant Rev protein with N terminal 6X histidine tag by induction with 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fermentas, Germany) for 6 hours at 37°C. Rev was purified under native conditions using Talon-affinity resin (Clontech, USA) according to the manufacturer’s protocol and eluted with 150 mM imidazole. After purification, Rev protein was dialyzed in modified Rev-buffer [46 (link)] with the composition of 50 mM sodium phosphate buffer, pH 7.0, 150 mM NaCI, 10 mM K2S04, and 1 mM DTT. The protein was concentrated using centricon with 3 kDa cutoff (Millipore, USA). Protein concentration was measured by 1X Bradford Dye Reagent (Bio-Rad, USA) according to the manufacturer’s instructions. The purified protein was checked on 15% SDS PAGE (Additional file 1 : Figure S1A) and confirmed by Western blot using 1:2000 dilution of mouse anti-Rev antibody (SantaCruz Biotechnology Inc., USA) and 1:2500 dilution of HRP conjugated goat anti-mouse IgG antibody (SantaCruz Biotechnology Inc., USA) followed by detection with Pierce ECL western blotting substrate (Thermo Scientific, USA) and visualized using VersaDoc gel imaging system (BioRad, USA).
7
Quantitative O-GlcNAc Detection Protocol
8
Protein Expression Analysis in Cells
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Total protein was extracted from collected cells in RIPA buffer (Sigma-Aldrich; Merck KGaA) containing 1 mM PMSF (Sigma-Aldrich; Merck KGaA) and total protein concentration was quantified using a BCA assay (Beyotime Institute of Biotechnology). In total, 20 µg proteins were separated by 10% SDS-PAGE and subsequently transferred onto a PVDF membrane (EMD Millipore), which was blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) diluted in TBS-0.1% Tween-20 buffer at room temperature for 2 h. The membranes were then incubated with the following primary antibodies (all 1:1,000) at 4˚C overnight: Anti-ARF6 (Abcam; cat. no. ab226389), anti-Rac1 (Abcam; cat. no. ab155938), anti-Bcl-2 (Abcam; cat. no. ab194583), anti-Bax (Abcam; cat. no. ab263897), anti-Erk1/2 (Abcam; cat. no. ab17942), anti-p-ERK1/2 (Abcam; cat. no. ab214362) and anti-β-actin (Abcam; cat. no. ab213262). Following the primary antibody incubation, the membranes were washed with TBST and incubated with a HRP-conjugated secondary antibody (Abcam; cat. no. ab6721; 1:5,000) at room temperature for 2 h. Protein bands were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.; cat. no. 34080) on a VersaDoc™ gel imaging system (Bio-Rad Laboratories, Inc.). Densitometric analysis was performed using the Image J software (version 1.51; National Institutes of Health).
9
Western Blot Analysis of γ-H2AX
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Treated cells were lysed with 5% sodium dodecyl sulfate (SDS) sample buffer containing protease inhibitor cocktail (Roche). Proteins were loaded to SDS polyacrylamide gel, were separated by electrophoresis, were transferred to polyvinylidene difluoride membrane, and were immunoblotted with antibodies against γ-H2AX (clone JBW301; Millipore) and α-tubulin. Primary antibodies were detected with HRP conjugated goat secondary antibody. The immunoblot was developed using the VersaDoc Gel Imaging System (BioRad).
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Treated cells were lysed with 5% sodium dodecyl sulfate (SDS) sample buffer containing protease inhibitor cocktail (Roche). Proteins were loaded to SDS polyacrylamide gel, were separated by electrophoresis, were transferred to polyvinylidene difluoride membrane, and were immunoblotted with antibodies against γ-H2AX (clone JBW301; Millipore) and α-tubulin. Primary antibodies were detected with HRP conjugated goat secondary antibody. The immunoblot was developed using the VersaDoc Gel Imaging System (BioRad).
10
Western Blot Analysis of Tau and CDK5 Proteins
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