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Recombinant RAD21 抗体 (N-Term)

Name: Recombinant RAD21 抗体 (N-Term)
SKU: ABIN6731038
Availability: OutOfStock
Rating: 5 (1 reviews)

RAD21 适用: 人, 小鼠 WB, IF 宿主: 小鼠 Monoclonal unconjugated Recombinant Antibody

Cleavage Under Targets and Release Using Nuclease validation image for anti-RAD21 Homolog (RAD21) (N-Term) antibody (ABIN6731038) (Recombinant RAD21 抗体 (N-Term))

CUT&RUN

Rad21 antibody (rAb) tested by Western blot. (Recombinant RAD21 抗体 (N-Term))

Rad21 antibody (rAb) tested by Immunofluorescence. (Recombinant RAD21 抗体 (N-Term))

4 images

(1 validation)

产品编号 ABIN6731038

发货至: 中国

Datasheet as PDF

抗原 See all RAD21 抗体

RAD21 (RAD21 Homolog (RAD21))

抗体类型

Recombinant Antibody
抗原表位
16

12

4

2

2

1

1

1

1

1

1

1

1
N-Term
适用
54

29

29

6

5

5

5

4

3

3

2

2

2

2

1
人, 小鼠
宿主
49

3

1
小鼠
Compatible Secondaries
克隆类型
46

8
单克隆
标记
32

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1
This RAD21 antibody is un-conjugated
应用范围
46

16

13

13

10

10

9

7

6

5

4

2

1

1

1
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

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RAD21 适用: 人, 小鼠 WB, IF, IP, IHC (p), ICC 宿主: 兔 Polyclonal unconjugated

anti-RAD21 Homolog (RAD21) antibody
RAD21 适用: 人 WB, IHC, IF, IP, ChIP 宿主: 兔 Polyclonal unconjugated

anti-RAD21 Homolog (RAD21) (AA 287-403) antibody
RAD21 适用: 人 WB, ELISA, IHC, FACS, ICC 宿主: 小鼠 Monoclonal 1B6D1 unconjugated

anti-RAD21 Homolog (RAD21) (C-Term) antibody
RAD21 适用: 人, 小鼠 WB, ELISA, IHC, IF, ICC 宿主: 兔 Polyclonal unconjugated

anti-RAD21 Homolog (RAD21) (AA 373-422) antibody
RAD21 适用: 人, 小鼠, 大鼠, Cow, 犬, 马, 兔, 豚鼠, 猴, 小鸡, Bat, Pig, 非洲爪蟾 WB 宿主: 兔 Polyclonal unconjugated

anti-RAD21 Homolog (RAD21) antibody
RAD21 适用: 人 WB, ChIP 宿主: 兔 Monoclonal unconjugated

anti-RAD21 Homolog (RAD21) (C-Term) antibody
RAD21 适用: 人, 小鼠, Hamster WB, IF 宿主: 兔 Polyclonal unconjugated

anti-RAD21 Homolog (RAD21) (C-Term) antibody
RAD21 适用: 人, 小鼠, 大鼠, Cow, 斑马鱼 WB, IHC, IF, IC 宿主: 兔 Polyclonal unconjugated

anti-RAD21 Homolog (RAD21) (AA 531-631) antibody
RAD21 适用: 人, 大鼠 WB, ELISA, FACS, IF (cc), IF (p), IHC (p), IHC (fro) 宿主: 兔 Polyclonal unconjugated

anti-RAD21 Homolog (RAD21) (AA 501-535) antibody
RAD21 适用: 人, 小鼠, 大鼠 WB 宿主: 兔 Polyclonal RB56184 unconjugated

应用备注

Validated Applications:
IF: 0.25 - 1 µg/ml
WB: 0.5- 2 μg/mL

说明

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
生效 #104617 (Cleavage Under Targets and Release Using Nuclease)

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.

Validation Images

Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using RAD21 ABIN6731038 (right) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (left).

1. Alignment tracks from CTCF CUT&RUN in HEK293T cells. 2. Alignment tracks from CUT&RUN targeting RAD21 in HEK293T cells using ABIN6731038 antibody showing the FBRS locus. 3. RefSeq Genes.
Full Methods
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.

Experimental Notes
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
抗原

RAD21 (RAD21 Homolog (RAD21))

别名

Rad21 (RAD21 产品)

分子量

100 kDa

途径

Positive Regulation of Endopeptidase Activity