Recombinant RAD21 抗体 (N-Term)
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- 抗原 See all RAD21 抗体
- RAD21 (RAD21 Homolog (RAD21))
- 抗体类型
- Recombinant Antibody
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抗原表位
- N-Term
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适用
- 人, 小鼠
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宿主
- 小鼠
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克隆类型
- 单克隆
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标记
- This RAD21 antibody is un-conjugated
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应用范围
- Western Blotting (WB), Immunofluorescence (IF)
- 品牌
- AbFlex®
- 纯化方法
- Protein A Chromatography
- 免疫原
- This antibody was raised against recombinant protein corresponding to the N-terminal half of mouse Rad21.
- 亚型
- IgG2a
- Top Product
- Discover our top product RAD21 Primary Antibody
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- 应用备注
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Validated Applications:
IF: 0.25 - 1 µg/ml
WB: 0.5- 2 μg/mL
- 说明
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AbFlex® antibodies are recombinant antibodies (rAbs) that have been generated using defined DNA sequences to produce highly specific, reproducible antibodies. Each AbFlex antibody contains a 6XHis tag, an avidin tag sequence for enzymatic biotin conjugation using the biotin ligase, BirA, and a sortase recognition motif (LPXTG) to attach a variety of labels directly to the antibody including fluorophores, enzymatic substrates (HRP, AP), peptides, drugs as well as solid supports. AbFlex Rad21 antibody was expressed as full-length IgG with mouse immunoglobulin heavy and light chains (IgG2a isotype) in mammalian 293 cells
- 限制
- 仅限研究用
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- by
- Cantù Lab, Gene Regulation during Development and Disease, Linköping University
- No.
- #104617
- 日期
- 2024.12.06
- 抗原
- RAD21
- Lot Number
- 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.
- Primary Antibody
- ABIN6731038
- 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.
- Remove the microcentrifuge tube from the magnetic stand.
- Repeat the wash twice for a total of three washes.
- Gently resuspend the ConA Beads in 44ul 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 incubates 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, 2mM EDTA)
- Incubate 5 min at RT.
- Place the tube on the magnet stand and when the liquid is clear remove the supernatant.
- Resuspend the beads in 200µl of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) x each sample.
- Primary antibody binding
- Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody.
- Add 2 µL antibody (RAD21 ABIN6731038, 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 ON.
- Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
- Remove the microcentrifuge tubes from the magnetic stand.
- 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.
- Remove tubes from the magnetic stand.
- Resuspend the beads in 200 µL of pAG-MNase premix.
- Incubate 30 min at 4 °C
- Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
- Remove the microcentrifuge tubes from the magnetic stand.
- 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.
- Always in ice, place the samples on the magnetic rack and when the liquid is clear remove the supernatant.
- Resuspend the samples in 100 µl of the 2 mM CaCl2 mix and incubate in ice for exactly 30 min.
- Place the sample on the magnet stand and when the liquid is clear remove the supernatant.
- 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)
- Take the Mag-Bind® TotalPure NGS beads (Omega Bio-Tek, M1378-01) from the storage and wait until they are RT.
- Add 2x volume of beads to each sample (e.g. 100 µL of beads for 50 µL of sample).
- Incubate the beads and the sample for 15 min at RT.
- During incubation prepare fresh EtOH 80%.
- Place the PCR tubes on a magnet stand and when the liquid is clear remove the supernatant.
- Add 200 µl of fresh 80% EtOH to the sample without disturbing the beads (Important!!! Do NOT resuspend the beads or remove the tubes from the magnet stand or the sample will be lost).
- Incubate 30 sec at RT.
- Remove the EtOH from the sample.
- Repeat the wash with 80% EtOH.
- Resuspend the beads in 25 µL of 10 mM Tris.
- Incubate the sample for 2 min at RT.
- Repeat the 2x beads clean up as described before (this time with 50 µlL of beads for each sample).
- Resuspend the beads + DNA 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 using bbTools bbduk (BBMap - Bushnell B. - sourceforge.net/projects/bbmap/) 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.
- Cell harvest and nuclear extraction
- Experimental Notes
生效 #104617 (Cleavage Under Targets and Release Using Nuclease)Validation ImagesFull Methods -
- 状态
- Liquid
- 缓冲液
- Purified IgG in 140 mM Hepes, pH 7.5, 70 mM NaCl, 32 mM NaOAc, 0.035 % sodium azide, 30 % glycerol.
- 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
- -20 °C
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- 抗原
- RAD21 (RAD21 Homolog (RAD21))
- 别名
- Rad21 (RAD21 产品)
- 分子量
- 100 kDa
- 途径
- Positive Regulation of Endopeptidase Activity
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