Ab5823

Manufactured by Abcam

Sourced in United States

Ab5823 is a primary antibody that specifically recognizes the target antigen. It is designed for use in various immunoassay applications.

Automatically generated - may contain errors

Lab products found in correlation

Ab110641, by Abcam (4 mentions) Ab130740, by Abcam (4 mentions) Trizol reagent, by Thermo Fisher Scientific (4 mentions) Ab22397, by Santa Cruz Biotechnology (2 mentions) Sc7824, by Abcam (2 mentions) P0493, by Merck Group (2 mentions) Ab49405, by Abcam (2 mentions) Confocal laser scanning microscope, by Zeiss (2 mentions) Ab13370, by Abcam (2 mentions) Ab22397, by Abcam (2 mentions) Bca protein assay, by Thermo Fisher Scientific (2 mentions) Anti prmt5, by Merck Group (2 mentions) Ab10158, by Abcam (1 mentions) E2f1 c20, by Santa Cruz Biotechnology (1 mentions) Ac 74, by Merck Group (1 mentions) Ab70774, by Abcam (1 mentions) Ab8580, by Abcam (1 mentions) Ab6002, by Abcam (1 mentions) H4r3me2a, by Active Motif (1 mentions) H4k5ac, by Merck Group (1 mentions)

17 protocols using ab5823

Western Blot Analysis of Protein Targets

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For immunoblots, cells were harvested in radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 1% (w/v) Igepal CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.2 mM sodium orthovanadate, and protease inhibitor cocktails] and incubated on ice for 30 min before SDS–polyacrylamide gel electrophoresis. The following antibodies were used in immunoblots: p100/TSN (A302-883A, Bethyl Laboratories; RRID: AB_10631268), E2F1 (C20, Santa Cruz Biotechnology; RRID: AB_631394), E2F1 (A300-766A, Bethyl Laboratories; RRID: AB_2096774), HA (16B12, Covance; RRID: AB_10063630), FLAG (M2, Sigma-Aldrich; RRID: AB_262044), β-actin (AC-74, Sigma-Aldrich; RRID: AB_476697), H4R3me2s (ab5823, Abcam; RRID: AB_10562795), histone H4 (ab10158, Abcam; RRID: AB_296888), and SENP7 (donated by R. Hay, University of Dundee, UK).

Roworth A.P., Carr S.M., Liu G., Barczak W., Miller R.L., Munro S., Kanapin A., Samsonova A, & La Thangue N.B. (2019). Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control. Science Advances, 5(6), eaaw4640.

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Chromatin Immunoprecipitation Assay for AR Promoter

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The LNCaP stable cell line or parental cells were cultured in the presence or absence of Dox (1 μg/ml) for 96 h. At the end of induction, 270 μl of 37% formaldehyde was added into each dish and incubated at room temperature for 10 min. Then 1 ml of 1.25 M glycine was added to stop the cross-linking reaction. Cells were then harvested, resuspended in 1 ml of immunoprecipitation buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 5 mM EDTA, 1% TritonX-100, 0.5% NP-40, 0.5 mM DTT and protease and phosphatase inhibitors), and finally sonicated (Branson Sonifier250set) to prepare sheared chromatin. Antibodies against PRMT5 (Millipore, 07-405), Sp1 (Santa Cruz, SC7824), Brg1 (Abcam, ab110641), H4R3me2s (Abcam, ab5823), H3R8me2s (Abcam, ab130740), H2AR3me2s (Abcam, ab22397), and IgG (Santa Cruz, SC2027) were used to immunoprecipiate protein-DNA complexes for isolation of PCR-ready DNA using the Fast ChIP protocol described previously ^{34 (link)}. The co-immunoprecipitated proximal promoter region of AR (−493 to −226) was quantified by qRT-PCR. Results were normalized to the IgG control and are presented as mean ± SD from three independent experiments. Student's t-test was used to determine the statistical significance.

Deng X., Shao G., Zhang H.T., Li C., Zhang D., Cheng L., Elzey B.D., Pili R., Ratliff T.L., Huang J, & Hu C.D. (2016). Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth. Oncogene, 36(9), 1223-1231.

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Chromatin Immunoprecipitation Validation Protocol

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ChIP assays were performed with H1299 cells in accordance with
standard protocols^{43 (link)}. Normal rabbit IgG served as the control. ChIP samples were
analyzed by quantitative real-time PCR using the FastStart Universal SYBR Green
Master (Roche). A standard curve was prepared for each set of primers using serial
titration of the input DNA. The percentage of ChIP DNA was calculated relative to
the input DNA from primer-specific standard curves using the Rotor-Gene 6000
Series Software 1.7. The primer sequences for ChIP are listed in Supplementary
Table 4. Antibodies used were: H4S1ph
(Abcam; ab14723), H4K5ac (Millipore; CS204381), H4R3me2a (Active Motif; 39705),
H4R3me2s (Abcam; ab5823), CK2α (Abcam; ab70774), H3K4me3 (Abcam; ab8580), and
H3K27me3 (Abcam; ab6002).

Ju J., Chen A., Deng Y., Liu M., Wang Y., Wang Y., Nie M., Wang C., Ding H., Yao B., Gui T., Li X., Xu Z., Ma C., Song Y., Kvansakul M., Zen K., Zhang C.Y., Luo C., Fang M., Huang D.C., Allis C.D., Tan R., Zeng C.K., Wei J, & Zhao Q. (2017). NatD promotes lung cancer progression by preventing histone H4 serine phosphorylation to activate Slug expression. Nature Communications, 8, 928.

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Western Blot Analysis of Protein Markers

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Cells were washed with PBS and lysed in lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, leupeptin). The concentration of the protein was quantified using the BCA protein assay (Thermo Fisher Scientific, USA). Western blotting was implemented in accordance with the standard methods [47 (link)]. The antibodies used were as follows: PRMT5 (P0493, Sigma), GAPDH (HRP-60004, Proteintech), CASP1 (3866, CST), cleaved-CASP1 (4199, CST), H4R3me2s (ab5823, Abcam), cleaved-CASP3 (9661, CST), N-GSDMD (ab215203, Abcam), IL-1b (66737-1-Ig, Proteintech), and IL-18 (10663-1-AP, Proteintech). Quantifications of all western blotting signals with 3 repeated experiments are shown in Supplemental Fig. 2A–D as histograms.

Xia T., Liu M., Zhao Q., Ouyang J., Xu P, & Chen B. (2021). PRMT5 regulates cell pyroptosis by silencing CASP1 in multiple myeloma. Cell Death & Disease, 12(10), 851.

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Histone Peptide Modification Assay

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The histone peptides were purchased from Chinese Peptide Company, all the primers and DNA substrates used in this paper was synthesized by GenScript, Inc. Four deoxynucleotide triphosphates (dNTPs) were purchased from New England Biolabs (N0446S). [γ-³²P]-ATP (BLU002A, 250 μCi) and [α-³²P]-dCTP (NEG513H, 250 μCi) were purchased from PerkinElmer. The M2 beads and M-280 beads were purchased from Sigma-Aldrich. The DNA Ligase I was purchased from Abnova. The antibodies used in this paper are as follow: anti-FEN1 (GTX70185, Genetex), anti-PRMT5 (sc-376937, Santa Cruz), anti-8-oxoG (sc-130914, Santa Cruz), anti-H4 (PTM-1003, PTM Bio), anti-H4R3me1 (ab17339, Abcam), anti-H4R3me2s (ab5823, Abcam), anti-OGG1 (ab135940, Abcam), anti-Flag (AP0007MH, Bioword), anti-GAPDH (AP0063, Bioworld), anti-Tubulin (AM031A, Abgent).

Ma Z., Wang W., Wang S., Zhao X., Ma Y., Wu C., Hu Z., He L., Pan F, & Guo Z. (2020). Symmetrical dimethylation of H4R3: A bridge linking DNA damage and repair upon oxidative stress. Redox Biology, 37, 101653.

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Western Blot Analysis of Protein Expression

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Cells were trypsinized, washed with phosphate- buffered saline (PBS) and lysed in Cell lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1% Triton X-100, Sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, leupeptin). Equal amounts of protein were separated by 8 ~ 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Roche). The membranes were blocked for 1 ~ 2 h at room temperature in PBST with 5% (w/v) non-fat milk and incubated overnight at 4°C with primary antibodies. After incubation with secondary antibodies, proteins were visualized with the ECL detection system (Thermo Fisher Scientific). The following antibodies were used: PRMT5 (P0493, Sigma), c-Myc (ab32072, ab56, Abcam), GAPDH (M171-3, MBL), PTEN (ab32199, Abcam), p21 (#2947, CST), p63 (ab124762, Abcam), p18 (ab192239, Abcam), p57 (ab75974, Abcam), HSP70 (#4873, CST), H4R3me2s (ab5823, Abcam) and Histone H4 (16047-1-AP, Proteintech).

Liu M., Yao B., Gui T., Guo C., Wu X., Li J., Ma L., Deng Y., Xu P., Wang Y., Yang D., Li Q., Zeng X., Li X., Hu R., Ge J., Yu Z., Chen Y., Chen B., Ju J, & Zhao Q. (2020). PRMT5-dependent transcriptional repression of c-Myc target genes promotes gastric cancer progression. Theranostics, 10(10), 4437-4452.

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Chromatin Immunoprecipitation for Epigenetic Regulation Analysis

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Cells (5 × 10⁶ cells per assay) were cross-linked with 1% formaldehyde (Sigma; F8775), quenched with 0.125 M glycine, and then sonicated using a Bioruptor UCD-300 (Diagenode) to yield chromatin fragments. Then 100 μg of lysate was immunoprecipitated with anti-IgG (negative control), anti-PRMT5 (Sigma; P0493), anti-H4R3me2s (Abcam: ab5823), or anti-H3R8me2s (Abcam; ab272149) antibodies immobilized on protein A-Sepharose beads (Sigma; P3391). The immunoprecipitated complexes were washed sequentially with washing buffer (10 mM Tris–HCl, 500 mM NaCl, 1% Trition X-100, 0.1% SDS, 0.5% Na-deoxycholate). After digestion with proteinase K (Beyotime; ST532), the DNA fragments were extracted with phenol:chloroform:isoamyl alcohol (25:24:1 v:v:v) and precipitated with 100% ethanol at -20 °C in the presence of 0.3 M sodium acetate (pH 5.2) and 2 µg of glycogen (Beyotime; D0812). Finally, the glycogen-protein pellet was suspended in H₂O and the purified DNA was subjected to real-time PCR analyses. The ChIP primer sequences are listed in Supplementary Table S4.

Yao B., Zhu S., Wei X., Chen M.K., Feng Y., Li Z., Xu X., Zhang Y., Wang Y., Zhou J., Tang N., Ji C., Jiang P., Zhao S.C., Qin C, & Feng N. (2022). The circSPON2/miR-331-3p axis regulates PRMT5, an epigenetic regulator of CAMK2N1 transcription and prostate cancer progression. Molecular Cancer, 21, 119.

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Western Blot Analysis of Protein Markers

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Cells were harvested and lysed in RIPA buffer. Proteins from lysed cells were separated from 8 to 12% SDS‐PAGE gels and transferred to 0.22 μm polyvinylidene difluoride membranes (Millipore, Frankfurt, Germany). Then, 5% milk in TBST was used to block nonspecific binding sites. The membranes were incubated with primary antibodies overnight at 4°C. Then, the blots were washed with 0.1% TBST three times and incubated with horseradish peroxidase‐conjugated antibodies. The protein was detected using SuperSignal West Dura (Thermo, Waltham, USA). The antibodies used for western blotting were against GAPDH (sc‐293335), PRMT5 (Abcam ab109451), BTG2 (Abcam ab85051), Cyclin D1 (Abcam ab134175), Cyclin E1 (sc‐377100), p44/42 MAPK (Erk1/2) (CST #4695), phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST #4370), phospho‐c‐Raf (Ser338) (CST #9427), Histone H4 symmetric dimethyl R3 (H4R3me2s) (Abcam ab5823), and Histone H3 (Abcam ab1791).

Jiang H., Zhu Y., Zhou Z., Xu J., Jin S., Xu K., Zhang H., Sun Q., Wang J, & Xu J. (2018). PRMT5 promotes cell proliferation by inhibiting BTG2 expression via the ERK signaling pathway in hepatocellular carcinoma. Cancer Medicine, 7(3), 869-882.

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Histone H4 and E2F2 Binding Assay

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Nuclear extracts were obtained from undifferentiated QCMCs according to the manufacturer’s protocol and used for EMSAs using LightShift Chemiluminescent EMSA Kit (Thermo Fisher Corp., Waltham, MA, USA) with modifications. Briefly, 200 fmol of 5′-biotin labeled histone H4 or E2F2 probes (listed in Table 1) was incubated with 10 μg nuclear extracts, 2 μL of 10 × binding buffer, 1 μL of poly dI·dC and 1 μL of 50% glycerol in a volume of 20 μL. For the competition assay, unlabeled or mutated probes were added to the reaction mixture for 10 min before adding the labeled histone H4 or E2F2 probes. Finally, 10 μg of histone H4 (ab5823, Abcam, Cambridge, MA, USA) or E2F2 (ab138515, Abcam) antibody was added to the reaction mixture for the super-shift assay. The DNA-protein complexes were separated on a 6% non-denaturing polyacrylamide gel and based on three independent experiments. Images were captured using the molecular imager ChemiDoc™ XRS+ system (Bio-Rad, Hercules, CA, USA).

Wei D., Li A., Zhao C., Wang H., Mei C., Khan R, & Zan L. (2018). Transcriptional Regulation by CpG Sites Methylation in the Core Promoter Region of the Bovine SIX1 Gene: Roles of Histone H4 and E2F2. International Journal of Molecular Sciences, 19(1), 213.

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Immunostaining of Meiotic Markers

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After rehydration and antigen retrieval, the 5 μm sections were incubated with 5% donkey serum in 0.3% triton X-100 for 1 hr. Then the sections were incubated with the primary antibodies for 1.5 hrs, and the corresponding TRITC-conjugated donkey anti-rabbit IgG (1:150, Jackson) and FITC-conjugated donkey anti-mouse IgG (1:150, Jackson) for 1.5 hrs at room temperature. The following dilutions of primary antibodies were used: PRMT5 (1:200, Millipore, 07-405), DAZL (1:100, AbD Serotec, MCA2336), STRA8 (1:200, Abcam, ab49405), SCP3 (1:200, Abcam, ab15093), γH2AX (1:400, Millipore, 05-636), DMC1 (1:100, Santa Cruz, sc-22768 ), H3R2me2s (1:50, Millipore, ABE460), H4R3me2s (1:100, Abcam, ab5823). After three times wash in PBS, the nuclei was stained with DAPI. The sections were examined with confocal laser scanning microscope (Carl Zeiss Inc., Thornwood, NY).

Wang Y., Zhu T., Li Q., Liu C., Han F., Chen M., Zhang L., Cui X., Qin Y., Bao S, & Gao F. (2015). Prmt5 is required for germ cell survival during spermatogenesis in mice. Scientific Reports, 5, 11031.

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