Nine-month-old P310S mice were injected IP with vehicle (PBS) or HJ8.5 of 10 or 50 mg/kg (n = 3/group). Forty-eight hours later, mice were sacrificed and plasma collected. A column was packed with 220 μL of protein A/G agarose resin (Thermo Scientific) and 50 μL plasma samples were 1:1 diluted with PBS and incubated on the column for 2.5 h at 4°C. The columns were then spun at 1000g for 1 min in a refrigerated microfuge 20R centrifuge (Beckman Coulter, Indianapolis, IN). The flow through contained free tau unbound to antibodies. The column was washed four times with PBS, then eluted with 0.1 mol/L glycine (pH 2.7), and eluates were neutralized with 1 mol/L Tris pH 9.0. Following elution, samples contained tau bound to antibodies. All the samples were stored at −80°C until the tau levels were measured by ELISA.
Protein a g agarose resin
Manufactured by Thermo Fisher Scientific
Protein A/G agarose resin is a chromatographic media designed for the purification of antibodies. It consists of recombinant Protein A and Protein G coupled to agarose beads. Protein A and Protein G are bacterial proteins with high affinity for the Fc region of immunoglobulins, enabling the efficient capture and purification of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Microfuge 20r centrifuge, by Beckman Coulter (1 mentions)
Anti flag, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Mouse igg1 isotype control, by BD (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
3 protocols using protein a g agarose resin
1
Quantifying Antibody-Bound Tau in Plasma
2
Antibody Characterization and Immunoprecipitation
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Commercial antibodies were BD34 (product number 610947; BD Biosciences, San Jose, CA); 2B1 (MA1-82452; ThermoFisher Scientific, Rockford, IL); mouse IgG1 isotype control (554121; BD Biosciences); anti-GFP (product number A6455; Invitrogen); anti-Flag (F7425; Invitrogen); Alexa Fluor 488-conjugated goat anti-rat IgG and goat anti-rabbit IgG (A21210 and A11034, respectively; Invitrogen); Alexa Fluor 594-conjugated goat anti-rat IgG (A21471, Invitrogen); horseradish peroxidase-conjugated goat anti-mouse IgG, anti-rabbit IgG, and anti-rat IgG (7076, 7074, and 7077, respectively; Cell Signaling Technology, Danvers, MA); and anti-6X-His tag (372900, Invitrogen) M2 Flag affinity resin was purchased from MilliporeSigma (Burlington, MA). TCEP, Lipofectamine 2000, and protein A/G agarose resin were obtained from ThermoFisher Scientific.
3
ChIP-qPCR Analysis of Hd1 Promoter
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ChIP assay was performed according to the previous report with little modification (Cui et al. 2017) (link). In brief, 1.5 g of 3-wk-old rice seedling leaves were cross-linked with 1%(m/v) formaldehyde. Nuclei were extracted with Honda buffer (0.44 M sucrose, 1.25% (m/v) Ficoll, 2.5% (m/v) Dextran 40, 10 mM MgCl 2 , 20 mM Hepes-KOH, pH7.4, 0.5% (v/v) Triton X-100, 1 mM PMSF, 1× cocktail). Nuclear lysis buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA, 1% (m/v) SDS, 1× cocktail) was used to isolate chromatin. After chromatin was sonicated, anti-GFP antibody (Abcam, Cat. ab290) and Protein A/G agarose resin (Thermo Fisher Scientific, Cat. Pierce 20421) were used to specifically immunoprecipitate chromatin fragments. After reverse crosslinking and DNA purification, qPCR was used to detect the Hd1 promoter enriched by GFP-HBP1 and GFP-POH1. Primers used to amplify the fragments of Hd1 promoter were listed in Supplemental Table S2.
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