For detection of AID Ramos cellines, RNA was extracted using the Absolutely RNA Microprep Kit (Agilent Technilogies) and 150 ng RNA was reverse transcribed using random hexamers (Applied Biosystems) and SuperScript III Reverse Transcriptase kit (Invitrogen). For mRNA gene expression assays, probes were purchased from Applied Biosystems: HPRT1: Hs02800695_m1, AICDA: Hs00757808_m1. Reactions were run on a 7500 Real-Time PCR system (Applied Biosystems) in duplicate. Values are represented as the difference in Ct values normalized to HPRT1 for each sample. For AID protein detection, cells were lysed in lysis buffer (50mM Tris, 1% NP-40, 2mM EDTA) including protease inhibitor (Roche). Total cell lysates were separated by SDS page, transferred to PDVF membranes, probed with mouse anti-AID (Invitrogen) and anti-mouse HRP (Cell Signaling) and detected by chemiluminescence (Amersham ECL Prime Western Blotting detection Reagent) using a GBox documentation system (Syngene). For quantification, blots were stripped with stripping buffer (Pierce) and reprobed with a mouse anti-β-Actin antibody (Sigma-Aldrich).
Gbox documentation system
Manufactured by Syngene
Sourced in United Kingdom
The GBox documentation system is a compact and versatile imaging platform designed for capturing high-quality images and documentation of laboratory samples, gels, and other biological specimens. The system features advanced optics, a high-resolution camera, and intuitive software for acquiring, analyzing, and archiving images.
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Lab products found in correlation
Mouse anti β actin antibody, by Merck Group (2 mentions)
Ecl prime western blotting detection reagent, by Syngene (2 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Pcr machine, by Eppendorf (1 mentions)
Kapa2g robust hotstart readymix pcr kit, by Roche (1 mentions)
Protein a g bead, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Mastercycler gradient thermocycler, by Eppendorf (1 mentions)
Dreamtaq green pcr master mix kit, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
Ab6721, by Vector Laboratories (1 mentions)
Pi 2000 1, by Vector Laboratories (1 mentions)
Horse anti mouse, by Vector Laboratories (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
7 protocols using gbox documentation system
1
Detection of AID in Ramos Cells
2
Lipid Binding Assay for Vaspin
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Hydrophobic membranes (spotted with 100 pmol of various lipid species per spot) or PtdIns arrays (with serial dilutions of different immobilized PtdIns ranging from 100 to 1.56 pmol/spot) were blocked with PierceTM Protein free (TBS) blocking buffer (Thermo Scientific, Waltham, MA, USA) at room temperature for 1 h. Then, membrane lipid strips were incubated with 1 µg/mL vaspin at room temperature for 1 h. Incubation with mouse anti-vaspin antibody VP63 (AdipoGen) was followed by anti-mouse antibody (CST#7076P2, Cell Signaling Technology, Danvers, MA, USA). Bound protein was detected via enhanced chemiluminescence using a Gbox documentation system (Syngene, Bangalore, India).
3
Microsatellite Genotyping of Genetic Diversity
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A total of 12 microsatellite labeled primers with high polymorphism information content (PIC) value (part of the 30 primers recommended by Food and agriculture organization of the United Nations [FAO]) were used in the polymerase chain reaction (PCR) process (primers sequence, annealing temperature, range of PCR product size, and label used were based on Agung [9 ]). The PCR reagent composition was as follows: KAPA2G Robust Hot Start Ready Mix PCR Kit (Kapa Biosystems, Cape Town, South Africa) (18 μL), forward and reverse labeled primers (200 ng/μL), nuclease free water, and DNA samples (5 to 30 ng/μL). The program in the PCR machine (Eppendorf, Hamburg, Germany) was set at 94°C; 5 min (1 cycle), 35 cycles consisting of three stages: i) 94°C; 30 s, ii) 51°C to 59°C; 30 s (depending on primers), and iii) 72°C; 30 s, followed by one cycle at 72°C; 5 min. The PCR products were then visualized by electrophoresis using 2% agarose gel and followed by SyBr staining and captured in GBOX documentation System (Syngene, Cambridge, UK). Multiplex DNA fragment analysis was conducted afterwards for allele identification in 1st BASE Laboratory, Malaysia.
4
Protocol for B Cell Lysis and Co-Immunoprecipitation
5
Amplification of Ovine BMPR1B Gene
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The PCR amplification of ovine BMPR1B gene was performed using a primer pair of Forward: 5′-GTC GCT ATG GGG AAG TTT GGA TG-3′ and Reverse: 5′-CAA GAT GTT TTC ATG CCT CAT CAA CAC GGT C-3′ (Wilson et al. 2001) [17 (link)]. According to that primer, the target sequence of BMPR1B gene is along 140 bp (Fig. 1 ). The PCR reaction was performed in a total volume 10 μL containing 1.2 μL of DNA template (2.18 ng/μL), 10 pmol/μL each of primer, 2 × of DreamTaq Green PCR mastermix kit (ThermoScientific, USA) and 3.6 μL of nuclease-free water. The PCR reaction was performed in a Mastercycler Gradient thermocycler (Eppendorf-Germany) with amplification program comprised of pre-denaturation (95 °C at 2 min); followed by 36 cycles of denaturation (95 °C at 30 s), annealing (56.6 °C at 1 min; 30 s), and extension (72 °C at 30 s); and final extension (72 °C at 2 min).![]()
Primer position (underline) in the exon 8 of ovine BMPR1B gene (GenBank: GQ863576.1) along 140 bp. A Boorola (G or Fec.B) allele was caused by the missence mutation of c.109A > G or p.Q36R (R)
6
Western Blot Protein Detection
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Cells were lysed with NP40 cell lysis buffer supplemented with protease inhibitor cocktail (P2714, Sigma-Aldrich). Cells were centrifuged for 15 min at 12,000g at 4°C. Supernatant was saved and kept frozen until further use. DC protein assay (Bio-Rad) was used to quantify protein. The cell lysate was placed in SDS sample buffer and run in a precasted gel at 100 V. Proteins were transferred into nitrocellulose using the trans-Blot turbo (Bio-Rad). Primary antibodies are described above. Secondary antibodies were goat anti-Rb horseradish peroxidase (1:10,000; Abcam, ab6721) and horse anti-mouse (VectorLabs, PI-2000-1). Detection was performed using SuperSignal Atto (Thermo Fisher Scientific) using a G box documentation system (Syngene).
7
Quantitative Western Blot Analysis of PTPN22
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