The CUT&Tag assay was performed following previously described protocols with some modifications [24 (link)]. Briefly, 10 μL of Concanavalin A-coated magnetic beads (Bangs Laboratories) were added to each sample and incubated at room temperature for 10 min. A 1:50 dilution of anti-FOSL2 (Immunoway, YT1739) or IgG control antibody (Millipore, #12-370) was added and incubated overnight at 4°C. The secondary antibody (Millipore, #AP132) was diluted 1:100 in dig wash buffer, and the cells were incubated at room temperature for 60 min. A 1:100 dilution of the pA-Tn5 adaptor complex was prepared in dig-med buffer (0.01% Digitonin; 20 mM HEPES pH 7.5; 300 mM NaCl; 0.5 mM spermidine; 1 × protease inhibitor cocktail) and incubated with cells at room temperature for 1 h. DNA was purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. A total of 21 μL of DNA was mixed with 2 μL of a universal i5 and a uniquely barcoded i7 primer. A volume of 25 μL of NEBNext HiFi 2× PCR Master mix was added and mixed. The sample was placed in a Thermocycler with a heated lid. Library clean-up was performed using XP beads (Beckman Counter), and sequencing was performed on the Illumina Novaseq 6000 using 150-bp paired-end sequencing.
Ap132
Manufactured by Merck Group
Sourced in United States
The AP132 is a laboratory centrifuge designed for general-purpose applications. It provides reliable and consistent performance for a variety of sample types and volumes. The core function of the AP132 is to separate components of a liquid mixture based on their different densities through the application of centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Sephadex g 25, by Merck Group (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (2 mentions)
Ab5320, by Merck Group (2 mentions)
Ap183, by Merck Group (2 mentions)
Rnalater, by Merck Group (2 mentions)
Control igg antibody, by Merck Group (1 mentions)
Concanavalin a coated magnetic beads, by Bangs Laboratories (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
C0130, by Merck Group (1 mentions)
Nbp2 12506, by Novus Biologicals (1 mentions)
5 protocols using ap132
1
FOSL2 Transcription Factor CUT&Tag Assay
2
Labeling Goat-anti-Rabbit Antibodies
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Goat-anti-Rabbit secondary antibody solution (2 mg/mL, Millipore, AP132) was adjusted to pH~8.3 with sodium bicarbonate solution. 50 μL 4 mg/mL 4-yne Sulfo-NHS ester in DMSO solution was added dropwise to 250 μL protein solution while stirring. Reaction was kept under gentle stirring at RT for 1 h. The labeled antibodies were purified using gel permeation chromatography with Sephadex® G-25 (Sigma, G25150). Sephadex® G-25 gel was first swelled in PBS buffer at 90°C for 1 h and settled down at room temperature. The gel was then exchanged with fresh PBS buffer and stored at 4 °C. For gel chromatography, the column (diameter ~1 cm and length >12 cm) was loaded with swelled gels and equilibrated with PBS buffer. The antibody solution was applied and eluted with PBS. The first band with light color for 4-yne conjugated antibodies was collected. The solution was then centrifuged briefly and the supernatant was concentrated using Amicon® Ultra Centrifugal Filters (Millipore, UFC501096) to a final concentration of ~2 mg/mL in PBS with 5 mM sodium azide and stored at -20 °C.
3
Labeling Goat-anti-Rabbit Antibodies
4
Isolation and Characterization of Tumor Cell Subpopulations
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Mouse endothelial cells were isolated from vehicle- and BGJ398-treated T241-Vector and T241–FGF-2 tumors to examine PDGF gene expression. Mouse pericytes were isolated from non-treated T241-Vector and -FGF-2 tumors to detect FGFR gene expression. Small pieces of tumor tissues were digested for 45 min at 37 °C with 0.15% collagenases 1 and 2 (Sigma-Aldrich; C0130, C6885). Single cells were stained on ice for 30 min with a rat anti-mouse CD31 antibody (553370; BD-Pharmingen) to bind to endothelial cells or a rabbit anti-mouse NG2 antibody (AB5320; Millipore) to bind to pericytes, followed by incubation for 15 min with a goat anti-rat Cy5 antibody (Millipore; AP183) or a goat anti-rabbit Cy5 antibody (AP132S; Millipore). Cells were washed with PBS and further incubated with anti-Cy5/Alexa Fluor 647 micro beads (130-091-395; Miltenyi Biotec) on ice for 10 min. Magnetic labeled cells were separated using magnetic columns and collected cells were stored in RNAlater (Sigma-Aldrich; R0901) at 4 or −20 °C until further use of gene expression study.
5
Isolation of Tumor Cell Subpopulations
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Tissue samples of PDGF‐B‐ or vector‐transfected LLC and T241 tumors were cut into small pieces and incubated with 1.5 mg/mL type I (17018029, Gibco) and 1.5 mg/mL II collagenase (17101015, Gibco) in PBS at 37°C for 40‐60 min. After filtering with a cell filter, cells were centrifugated at 1500 rpm for 10 min. Cell pellets were collected and incubated with a rabbit anti‐mouse NG2 antibody (1:50, AB5320, Millipore), a rat anti‐mouse CD31 antibody (1:50, 553370, BD Pharmingen, San Diego, CA, USA), a rat anti‐mouse PDGFRα antibody (1:50, 14‐1401‐82, eBioscience), a rat anti‐mouse PDGFRβ antibody (1:50, 14‐14012‐82, eBioscience), and a rat anti‐F4/80 antibody (1:200, NBP2‐12506, Novus Biologicals, Littleton, CO, USA), followed by species matched antibody incubation in ice, including a Cy5‐labeled goat anti‐rabbit antibody (1:200, AP132S; Millipore) or Cy5‐labeled goat anti‐rat antibody (1:200, AP183, Millipore) for 30 min. Cells were further washed and incubated with anti‐Cy5/Alexa Fluor 647‐coated microbeads (130‐091‐395, Miltenyi Biotec) for 15 min, and subsequently subjected to magnetic separation through magnetic columns. Collected positive cells were used for further study or stored in RNAlater (R0901, Sigma‐Aldrich, Burlington, MA, USA) at ‐70°C until further use.
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