Ab21869

Manufactured by Abcam

Sourced in United Kingdom, Japan

Ab21869 is a purified recombinant antibody produced in HEK293 cells. The core function of this product is to serve as a research tool for targeted protein detection and analysis.

Automatically generated - may contain errors

Lab products found in correlation

Ab13840, by Abcam (2 mentions) Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions) Ts2 s sm, by Nikon (1 mentions) Lsm700 upright confocal microscope, by Zeiss (1 mentions) A11008, by Thermo Fisher Scientific (1 mentions) Ab1790, by Abcam (1 mentions) Anti β tubulin, by Merck Group (1 mentions) Vectorshield mounting media with dapi, by Vector Laboratories (1 mentions) A21429, by Thermo Fisher Scientific (1 mentions) Fluoview fv1000 confocal microscope, by Olympus (1 mentions) Histovt one, by Nacalai Tesque (1 mentions) Lsm 5 pascal, by Zeiss (1 mentions)

7 protocols using ab21869

Immunohistochemical Analysis of PIWI Protein in Femur Sections

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Longitudinal sections of femurs were transferred to glass slides and allowed to dry at room temperature, and incubated with primary antibodies directed against PIWI protein (Abcam, UK; ab21869; 1:200), overnight at 4 °C. Then the sections were incubated with secondary antibodies at 37 °C for 1 h and stained with 3′-diaminobenzidine (DAB). All the slides were observed and imaged via microscopy (TS2-S-SM, Nikon, Japan).
Longitudinal sections of femurs were transferred to glass slides and allowed to dry at room temperature. The sections were blocked with 10% normal goat serum for 30 minutes at room temperature and incubated with primary antibody working solution at 4 °C overnight. Then, sections were rinsed three times with phosphate-buffered saline and incubated with cyanine (cy3)-conjugated goat anti-rabbit IgG antibody in blocking buffer for 30 minutes at room temperature. The nuclei were counterstained with DAPI. Finally, the specimens were observed and imaged using a Leica DMi8 microscope (Carl Zeiss MicroImaging GHBH; Jena, Germany).

Chen G., Wang S., Long C., Wang Z., Chen X., Tang W., He X., Bao Z., Tan B., Lu W.W., Li Z., Yang D., Xiao G, & Peng S. (2021). PiRNA-63049 inhibits bone formation through Wnt/β-catenin signaling pathway. International Journal of Biological Sciences, 17(15), 4409-4425.

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Immunofluorescence Imaging of Piwil4 in MDAMB231 Cells

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MDAMB231 cells on gelatin-coated coverslips were fixed in 4% PFA and blocked before incubation with anti-Piwil4 (Abcam ab21869) followed by Cy3-AffiniPure Donkey anti-Rabbit IgG, and visualisation on a Carl Zeiss LSM 700 Upright Confocal microscope. DNA was stained with Hoechst-33342.

Keam S.P., Young P.E., McCorkindale A.L., Dang T.H., Clancy J.L., Humphreys D.T., Preiss T., Hutvagner G., Martin D.I., Cropley J.E, & Suter C.M. (2014). The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells. Nucleic Acids Research, 42(14), 8984-8995.

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Germ Cell Immunofluorescence and Isolation

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Anti‐MIWI2 primary antibody (ab21869, Abcam) and goat anti‐rabbit Alexa fluor 488 secondary antibody (A11008, Thermo Fisher Scientific) were used for immunofluorescence staining. Anti‐MILI antibody (PM044, MBL) and anti‐MIWI antibody (2C12, Wako) were used for immunoprecipitation. The affinity‐purified anti‐Mili26F antibody recognizing both MILI and MIWI 14 was used for Western blotting. PE anti‐mouse CD326 (Ep‐CAM) (G8.8, BioLegend) was used for germ cell sorting.

Nishimura T., Nagamori I., Nakatani T., Izumi N., Tomari Y., Kuramochi‐Miyagawa S, & Nakano T. (2018). PNLDC1, mouse pre‐piRNA Trimmer, is required for meiotic and post‐meiotic male germ cell development. EMBO Reports, 19(3), e44957.

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Fractionation and Western Blot of MDAMB231 Cells

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MDAMB231 cells (∼10 × 106) were pelleted by centrifugation at 4°C. Cytosolic proteins were recovered by lysing the cell pellet in 400 μl Buffer A (10 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM dTT, with protease inhibitor cocktail) for 15 min on ice before addition of 40 μl of 1% NP-40. The cell lysate was then centrifuged at 13 000 × g and the supernatant collected as the cytoplasmic fraction. The pellet was washed twice with 500 μl Buffer A with centrifugation at 13 000 × g, then lysed in 150 μl Buffer C (50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 137.5 mM NaCl, 10% Glycerol, 5 mM EDTA, 0.5% SDS, with protease inhibitor cocktail). The nuclear lysate was sonicated and cleared by centrifugation at 17 000 × g for 15 min. Separation of the cytoplasmic and nuclear fractions was verified by western blot for β-tubulin (anti- β-tubulin, Sigma T5201) and histone H2b (anti-H2B, Abcam ab1790). Western blot for Hiwi2 used the anti-Piwil4 antibody (Abcam ab21869). Western blot secondary antibodies were fluorescently labelled and membranes were visualised on a Licor Odyssey.

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Immunohistochemical Analysis of Testis

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Testes were fixed in 4% PFA in PBS overnight at 4 °C and embedded in paraffin. About 5 μm sections were cut, dewaxed, and rehydrated. Antigen retrieval was performed by microwaving the sections in 0.01 M sodium citrate buffer (pH 6.0). After rinsing with PBS, tissue sections were blocked in 5% normal goat serum (NGS) for 30 min. Testis sections were then incubated with anti-MIWI (1:50; 2079, Cell Signaling Technology), anti-MILI (1:100; PM044, MBL), anti-TDRKH (1:100; 13528-1-AP, Proteintech), anti-AIF (1:100; 5318, Cell Signaling Technology), anti-CRISP2 (1:100; 19066-1-AP, Proteintech), anti-MVH (1:100; ab13840, Abcam), anti-GASZ (1:50; 21550-1-AP, Proteintech), anti-LINE1 ORF1 (1:800), anti-MIWI2 (1:50; ab21869, Abcam), or FITC-conjugated mouse anti-γH2AX (1:500; 16–202A, Millipore) in 5% NGS at 37 °C for 2 h. After washing with PBS, sections were incubated with Alexa Fluor 555 goat anti-rabbit IgG (1:500; A21429, Life Technologies) for 1 h and mounted using Vectorshield mounting media with DAPI (H1200, Vector Laboratories) after washing. Fluorescence microscopy was performed using Fluoview FV1000 confocal microscope (Olympus, Japan).

Ding D., Liu J., Dong K., Midic U., Hess R.A., Xie H., Demireva E.Y, & Chen C. (2017). PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice. Nature Communications, 8, 819.

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Immunofluorescence Analysis of Mouse Testis

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Mouse testes were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4 °C and embedded in paraffin. Testis sections were cut at 5 μm, dewaxed and rehydrated. Antigen retrieval was performed in Tris-EDTA buffer (pH 9.0) or sodium citrate buffer (pH 6.0). Testis sections were blocked in 5% normal goat serum (NGS) at room temperature (RT) for 30 min. Testis sections were then incubated with anti-MIWI (1:100; 2079, Cell Signaling Technology), anti-MILI (1:100; PM044, MBL), anti-TDRKH (1:100; 13528–1-AP, Proteintech), anti-LINE1 ORF1 (1:800), anti-MIWI2 (1:50; ab21869, Abcam) or FITC-conjugated mouse anti-γH2AX (1:500; 16–202A, Millipore) in 5% NGS at 37 °C for 2 h. After washing with PBS, sections were incubated with Alexa Fluor 555 goat anti-rabbit IgG (1:500; A21429, Thermo Fisher Scientific) at RT for 1 h and mounted using Vectashield mounting media with DAPI (H-1200, Vector Laboratories). Fluorescence was photographed using Fluoview FV1000 confocal microscope.

Wei C., Yan X., Mann J.M., Geng R., Xie H., Demireva E.Y., Sun L., Ding D, & Chen C. (2023). PNLDC1 catalysis and postnatal germline function are required for piRNA trimming, LINE1 silencing, and spermatogenesis in mice. bioRxiv.

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Immunofluorescence Analysis of Morc3 in Testis

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Testes of the Morc3 homozygous and heterozygous mutant and wild type male mice were dissected and fixed in 4% paraformaldehyde for 2 h at 4°C (for D30 after birth) or 2% paraformaldehyde for 1 h at 4°C (for E14.5, 16.5, and 17.5 testes). After washing in PBS containing 10% and 20% sucrose, the testes were embedded in OCT compound. The cryosections blocked with 10% normal goat serum and 3% BSA in PBS for 0.5 h at room temperature were immunofluorescence stained, after treatment with HistoVT one (Nacalai Tesque Inc., Kyoto, Japan) for E16.5 and E17.5 testes. Sections were treated with anti-MORC3 antibody (D238-3; MBL, Japan), anti-MVH antibody (ab13840; abcam), anti-MILI antibody (#5940; Cell Signaling technology), anti-MIWI2 antibody (ab21869; abcam), or anti-MIWI polyclonal antibody (#2079; Cell Signaling) overnight at 4°C. Alexa Fluor 488-or 568-conjugated anti-rabbit immunoglobulins (H+L) or Alexa Fluor 488-or 568-conjugated anti-mouse immunoglobulins (H+L) (Molecular Probes, Eugene, OR, USA) were used as the secondary antibody for 1 h at room temperature.
Nuclei were counterstained with 1 μg/mL DAPI. Immunostained cryosections were examined under a confocal microscope (LSM5Pascal, Carl Zeiss Co. Ltd).

Nakano T., Kuramochi‐Miyagawa S., Nakayama M., Koseki H., Jacobsen S.E., & Kojima‐Kita K. (2020). MORC3, a novel MIWI2 association partner, is an epigenetic regulator of piRNA-dependent transposon silencing.

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