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豚鼠 anti-兔 IgG (Heavy & Light Chain) Antibody - Preadsorbed

ELISA, IHC, WB, CUT&Tag, CUT&RUN Polyclonal IgG unconjugated
Pubmed (105 references) Independent Validation (2 validations)
产品编号 ABIN101961
发货至: 中国
  • Key Features
    • 95+ publication references, 1 independent validation
    • Frequently used as CUT&RUN IgG negative control and CUT&Tag secondary antibody
    • Used in CUT&RUN and CUT&Tag protocols, e.g. Henikoff et al. (2018) PMID 29651053, Kaya-Okur et al. (2019, 2020) PMID 31036827 and PMID 32913232
    抗原 See all IgG products
    IgG
    抗原表位
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    • 424
    • 207
    • 132
    • 129
    • 95
    • 29
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    • 28
    • 22
    • 9
    • 6
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    • 1
    Heavy & Light Chain
    适用
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    • 699
    • 601
    • 477
    • 403
    • 243
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    • 176
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    • 138
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    • 2
    • 2
    • 1
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    兔
    宿主
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    • 572
    • 241
    • 112
    • 111
    • 33
    • 30
    • 21
    • 19
    • 15
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    豚鼠
    克隆类型
    • 3434
    • 175
    • 1
    多克隆
    应用范围
    • 1952
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    ELISA, Immunohistochemistry (IHC), Western Blotting (WB), Cleavage Under Targets and Tagmentation (CUT&Tag), Cleavage Under Targets and Release Using Nuclease (CUT&RUN)
    原理

    The Guinea Pig anti-Rabbit IgG antibody ABIN101961 is well suited as a CUT&RUN IgG negative control and as a secondary antibody in CUT&Tag. It is a component of all our CUT&RUN Product Sets.

    Find more products for CUT&RUN and CUT&Tag:

    特异性
    Rabbit IgG (H&L)
    交叉反应 （详细）
    No reaction was observed against Human, Mouse and Goat Serum Proteins.
    纯化方法
    Preadsorption: Solid phase absorption
    过滤
    Sterile filtered
    免疫原
    whole molecule of rabbit IgG
    亚型
    IgG
  • 应用备注
    The guinea pig anti-rabbit IgG antibody ABIN101961 is suitable for use in ELISA, immunohistochemistry, and Western Blot, CUT&RUN and CUT&Tag. Specific conditions for each assay should be optimized by the end user. General ABIN101961 dilution recommendations for different applications are as follows:
    • ELISA: 1:20,000 - 1:100,000
    • WB: 1:2,000 - 1:10,000
    • IHC: 1:1,000 - 1:5,000
    • CUT&RUN: 1:100
    • CUT&Tag: 1:100
    说明

    ABIN101961 is tested via ELISA to ensure that the titer against the antigen (Rb IgG) is above a certain threshold. We also test to make sure the titer against potentially cross-reactive human IgG, goat IgG, and mouse IgG is below a certain threshold.
    In addition, we test ABIN101961 against anti-guinea pig Serum, rabbit IgG, and rabbit serum in an immunoelectrophoresis assay.

    限制
    仅限研究用
  • 生效 #104174 (Cleavage Under Targets and Tagmentation)
    '独立验证'标志
    by
    Tom Taghon’s lab, Vakgroep Diagnostische Wetenschappen, Universiteit Gent
    No.
    #104174
    日期
    2019.12.18
    抗原
    rabbit IgG
    Lot Number
    43586
    Method validated
    Cleavage Under Targets and Tagmentation
    Positive Control

    rabbit anti-H3K27me3 monoclonal antibody

    Negative Control

    rabbit normal IgG antibody

    Notes

    Passed. ABIN101961 successfully increased the number of protein A binding sites in a CUT&Tag protocol on human primary thymocytes and PER-117 cells using a monoclonal rabbit H3K27me3 primary antibody.

    '独立验证'标志
    Validation Images
    CUT&Tag library generated from thymocytes using a monoclonal rabbit anti-H3K27me3 primary antibody and ABIN101961 as a secondary antibody. CUT&Tag library generated from thymocytes using a monoclonal rabbit anti-H3K27me3 primary antibody and ABIN101961 as a secondary antibody.
    CUT&Tag library generated from PER-117 cells using a monoclonal rabbit anti-H3K27me3 primary antibody and ABIN101961 as a secondary antibody. CUT&Tag library generated from PER-117 cells using a monoclonal rabbit anti-H3K27me3 primary antibody and ABIN101961 as a secondary antibody.
    Full Methods
    Primary Antibody
    rabbit IgG anti-H3K27me3 antibody (Cell Signaling Technology, 9733T, lot 14)
    Secondary Antibody
    ABIN101961
    Full Protocol
    • Profiling of H3K27me3 signals in human primary CD34+ sorted thymocytes and PER-117 cell line (50,000 cells each).
    • Carry out the CUT&Tag protocol according to the single-tube bench top protocol for CUT&Tag developed by Steven Henikoff’s lab as outlined on the protocols.io platform.
    • Use reagents as suggested in the original protocol. The pA-Tn5 fusion protein used in this validation was a courtesy of Steven Henikoff’s lab and used at 1:1000 dilution for 100 µL per sample.
    • Primary antibody binding with
      • 1µl/sample monoclonal rabbit IgG anti-H3K27me3 antibody (Cell Signaling Technology, 9733T, lot 14) or
      • 1µl/sample rabbit normal IgG (Cell Signaling, 2729S, lot 9).
    • Secondary antibody binding with 1µl/sample guinea pig anti rabbit antibody (antibodies-online, ABIN101961, lot 43586).
    • Determine library concentration on a Qubit Fluorometer using Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific, Q32581).
    • Determine library size distribution of all samples on an Agilent Fragment Analyzer using High Sensitivity Small DNA Fragment Analysis Kit (Agilent, DNF-477-0500).
    Experimental Notes

    • ABIN101961 successfully increased the number of protein A binding sites for each bound rabbit anti-H3K27me3 antibody in the human primary thymocytes and PER-117 cell line. This resulted in quantifiable amounts of tagmented genomic fragments after PCR amplification that showed a ladder-like distribution.

    • Library concentrations as measured on a Qubit Fluorometer were 0.122ng/µl and 0.538ng/µl for thymocytes and PER-117 cells respectively using a rabbit H3K27me3 primary antibody. Library concentration for both sample was below the instrument’s detections limit using the rabbit normal IgG primary antibody.

    • As discussed at the protocols.io, it is expected to observe a ladder-like distribution for H3K27me3 profiling. However, using lower cell numbers could alter the nucleosomal patterns and result in an increase in larger fragments. This is observed by Steven Henikoff’s lab as well. Nevertheless, it does not affect the quality of the sequencing results.

  • 生效 #104622 (Cleavage Under Targets and Release Using Nuclease)
    '独立验证'标志
    by
    Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104622
    日期
    2024.02.10
    抗原
    IgG
    Lot Number
    48661
    Method validated
    Cleavage Under Targets and Release Using Nuclease
    Positive Control

    Anti H3K4me (antibodies-online, ABIN3023251)

    Negative Control

    Guinea Pig anti-rabbit IgG (antibodies-online, ABIN101961)

    Notes

    Passed.

    '独立验证'标志
    Validation Images
    Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using ABIN6923144 (left) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (right). Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using ABIN6923144 (left) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (right).
    Alignment tracks from CUT&RUN in human fibroblast cells using ABIN101961 IgG antibody showing the WT1. 2. RefSeq Genes. Alignment tracks from CUT&RUN in human fibroblast cells using ABIN101961 IgG antibody showing the WT1. 2. RefSeq Genes.
    Full Methods
    Primary Antibody
    ABIN101961
    Secondary Antibody
    Full Protocol
    • Cell harvest and nuclear extraction
      • Harvest 250,000 HEK293T cells per antibody
      • Centrifuge cell solution 5 min at 600 x g at RT.
      • Remove the liquid carefully.
      • Gently resuspend cells in 2 mL of Nuclear Extraction Buffer (20 mM HEPES-KOH pH 8.2, 20% Glycerol, 0.05% IGEPAL, 0.5 mM Spermidine, 10 mM KCl, Roche Complete Protease Inhibitor EDTA-free).
      • Move the solution to a 2 mL centrifuge tube.
      • Pellet the nuclei 800 x g for 5 min.
      • Repeat the NE wash twice for a total of three washes.
      • Resuspend the nuclei in 40 µL NE Buffer per sample.
    • Concanavalin A beads preparation
      • Prepare one 2 mL microcentrifuge tube.
      • Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6923139).
      • Pipette 10 µL Con A Beads slurry for each sample into the 2 mL microcentrifuge tube.
      • Place the tube on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tube from the magnetic stand.
      • Pipette 1 mL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) into the tube and resuspend ConA beads by gentle pipetting.
      • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
      • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Repeat the wash twice for a total of three washes.
      • Gently resuspend the ConA Beads in 44 µL Binding Buffer.
    • Nuclei immobilization – binding to Concanavalin A beads
      • Carefully vortex the nuclei suspension and add 44 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
      • Close tube tightly and incubate 10 min at 4 °C.
      • Put the 2 mL tube on the magnet stand and when the liquid is clear, remove the supernatant.
      • Resuspend the beads in 1 mL of EDTA wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free, 2 mM EDTA).
      • Incubate 5 min at RT.
      • Resuspend the beads in 200 µL of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free).
    • Primary antibody binding
      • Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody.
      • Add 2 µL antibody (anti-H3K4me positive control ABIN3023251, and guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
      • Incubate at 4 °C overnight.
      • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
      • Repeat the wash five times for a total of six washes.
    • pAG-MNase Binding
      • Prepare a 1.5 mL microcentrifuge tube containing 200 µL of pAG mix per sample (200 µL of wash buffer + 120 ng pAG-MNase per sample).
      • Place the PCR tubes with the sample on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Resuspend the beads in 200 µL of pAG-MNase premix.
      • Incubate 30 min at 4 °C.
      • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
      • Repeat the wash five times for a total of six washes.
      • Resuspend in 100 µL of Wash Buffer.
    • MNase digestion and release of pAG-MNase-antibody-chromatin complexes
      • Place PCR tubes on ice and allow to chill.
      • Prepare a 1.5 mL microcentrifuge tube with 102 µL of 2 mM CaCl2 mix per sample (100 µL Wash Buffer + 2 µL 100 mM CaCl2) and let it chill on ice.
      • Resuspend the sample in 50 µL of 1x Urea STOP Buffer (8.5 M Urea, 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 0.5% IGEPAL).
      • Incubate the samples 1h at 4 °C.
      • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 200 µL PCR tubes.
    • DNA Clean up (Mag-Bind® TotalPure NGS - M1378-01)
      • Add 2x volume of beads to each sample (e.g. 100 µL of beads for 50 µL of sample).
      • Wash with 80% EtOH.
      • Resuspend the beads in 20 µL of 10 mM Tris.
    • Library preparation and sequencing
      • Prepare Libraries using KAPA HyperPrep Kit using KAPA Dual-Indexed adapters according to protocol.
      • Sequence samples on an Illumina NextSeq 500 sequencer, using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles), 36bp PE.
    • Peak calling
      • Trim reads using bbTools bbduk to remove adapters, artifacts, and repeat sequences.
      • Aligned reads were mapped to the hg38 human genome using bowtie with options -m 1 -v 0 -I 0 -X 500.
      • Use SAMtools to convert SAM files to BAM files and remove duplicates.
      • Use BEDtools genomecov to produce Bedgraph files.
      • Call peaks using SEACR with a 0.001 threshold and the option norm stringent.
    Experimental Notes
  • 状态
    Liquid
    浓度
    1.02 mg/mL
    缓冲液
    0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 0.01 % (w/v) NaN3, no stabilizer
    储存液
    Sodium azide
    注意事项
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    储存条件
    4 °C,-20 °C
    有效期
    12 months
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    Zhou, Halstead, Bonnet-Garnier, Schultz, Ross: "Histone remodeling reflects conserved mechanisms of bovine and human preimplantation development." in: EMBO reports, Vol. 24, Issue 3, pp. e55726, (2023) (PubMed).

    Li, Gao, Zhao, Guan, Morris, Finkelman, Huang: "The Hdc GC box is critical for Hdc gene transcription and histamine-mediated anaphylaxis." in: The Journal of allergy and clinical immunology, (2023) (PubMed).

    Boileau, Chen, Blelloch: "Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation during embryonic stem cell differentiation." in: Genome biology, Vol. 24, Issue 1, pp. 41, (2023) (PubMed).

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    Nguyen, Watson, Ortega, Woodruff, Jordan, Iwanaga, Yamamoto, Bailey, Jeong, Guntupalli, Behbakht, Gbaja, Arnoult, Chuong, Bitler: "Combinatory EHMT and PARP inhibition induces an interferon response and a CD8 T cell-dependent tumor regression in PARP inhibitor-resistant models." in: bioRxiv : the preprint server for biology, (2023) (PubMed).

    Buyukcelebi, Chen, Abdula, Duval, Ozturk, Seker-Polat, Jin, Yin, Feng, Wei, Bulun, Yue, Adli: "Engineered MED12 mutations drive uterine fibroid-like transcriptional and metabolic programs by altering the 3D genome compartmentalization." in: Research square, (2023) (PubMed).

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  • 抗原
    IgG
    Abstract
    IgG 产品
    物质类
    Antibody
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