Anti-RBPJK/RBPJ Picoband® Antibody

Western blot analysis of RBPJ using anti-RBPJ antibody (A00767-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates,
Lane 2: rat NRK whole cell lysates,
Lane 3: mouse NIH3T3 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RBPJ antigen affinity purified polyclonal antibody (Catalog # A00767-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for RBPJ at approximately 60KD. The expected band size for RBPJ is at 56KD.

Notch activity promotes microglial development in vivo and in vitro. a , b Immunofluorescence ( a ) and fluorescent intensity emission values ( b ) of RBPJ (red) in F4/80 + (green) cells in the sagittal sections of E14.5 Rbpj fl/fl and Cx3cr1 cre/+ Rbpj fl/fl mice ( n = 3 mice in each group). Each dot in ( b ) denotes one cell. Ten cells per mouse were quantified. The white arrow indicates the co-localized signals of RBPJ and F4/80, whereas white arrowhead point to the limited signals of RBPJ. c , d Immunofluorescence ( c ) and quantified cell density ( d ) of IBA1 + cells in the sagittal sections of E14.5 Rbpj fl/fl , Cx3cr1 cre/+ and Cx3cr1 cre/+ Rbpj fl/fl mice ( n = 6 mice in each group). Each dot denotes one mouse. Three slices per mouse were quantified. e Transcriptional levels of Sall1 , Tgfb1 , Fcrls , Slc2a5 , and Gpr34 in CD11b hi CD45 lo cells from E14.5 Rbpj fl/fl and Cx3cr1 cre/+ Rbpj fl/fl mice mesencephalon. The data are from three independent experiments. Each dot represents an independent experiment. f Immunofluorescence of IBA1 and Hes1 in the embryonic microglia in vitro furnished with MCSF or additional DLL3 for 5 days. g Quantification of IBA1 + cell density in ( f ). Data were pooled from three independent experiments. Cultured IBA1 + cells in each experiment were from twenty E12.5 mice mesencephalon. Each dot represents individual experiment. h The percentage of Hes1 - IBA1 + or Hes1 + IBA1 + cells after cultured 5 days in ( f ). The number in each histogram indicates the average percentage. Data are pooled from three independent experiments. i Transcriptional levels of Hes1, Mcm5, Dab2 , Pros1 , Tmem119 , Gpr34 , and Sall1 in embryonic microglia after cultured for 5 days. The data were from three independent experiments. Each dot represents an independent experiment. j Schematic diagram of Notch activation in mouse microglial differentiation, created with BioRender.com. Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns no significant, Unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.
Index in PubMed under a CC BY license. PMID: 36253375

Flow Cytometry analysis of HL-60 cells using anti-RBPJ antibody (A00767-1).
Overlay histogram showing HL-60 cells stained with A00767-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-RBPJ Antibody (A00767-1, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IF analysis of RBPJK/RBPJ using anti-RBPJK/RBPJ antibody (A00767-1).
RBPJK/RBPJ was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-RBPJK/RBPJ Antibody (A00767-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.