TCF7 抗体 (N-Term)
-
- 抗原 See all TCF7 抗体
- TCF7 (Transcription Factor 7 (T-Cell Specific, HMG-Box) (TCF7))
-
抗原表位
- N-Term
-
适用
- 人
-
宿主
- 兔
-
克隆类型
- 多克隆
-
标记
- This TCF7 antibody is un-conjugated
-
应用范围
- Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), Cleavage Under Targets and Release Using Nuclease (CUT&RUN)
- 特异性
- Recognizes endogenous levels of TCF7 protein
- 交叉反应 (详细)
- Bovine
- 产品特性
- Purified Polyclonal TCF7 antibody
- 纯化方法
- TCF7 antibody was purified by immunogen affinity chromatography
- 免疫原
- TCF7 antibody was raised in Rabbit using a KLH-conjugated synthetic peptide encompassing a sequence within the N-term region of human TCF7 as the immunogen
-
-
- 应用备注
-
The rabbit anti-TCF7 antibody ABIN5620945 is suitable for use in CUT&RUN, immunohistochemistry, immunoprecipitation, and Western Blot. Specific conditions for each assay should be optimized by the end user. General ABIN5620945 dilution recommendations for different applications are as follows:
IHC: 1:100-1:200
IP: 1:10-1:100
WB: 1:500-1:1,000
CUT&RUN: 1:100 - 限制
- 仅限研究用
-
- by
- Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
- No.
- #104349
- 日期
- 2022.02.28
- 抗原
- TCF7
- Lot Number
- X21031510
- Method validated
- Cleavage Under Targets and Release Using Nuclease
- Positive Control
Recombinant anti-H3K27me3 CUT&RUN Positive Control antibody (antibodies-online, ABIN6923144)
- Negative Control
Polyclonal guinea Pig anti-rabbit IgG (antibodies-online, ABIN101961)
- Notes
Passed. ABIN5620945 allows for TCF7 targeted digestion using CUT&RUN in human HEK293T cells.
- Primary Antibody
- ABIN5620945
- Secondary Antibody
- Full Protocol
- Cell harvest and nuclear extraction
- Harvest 250,000 HEK293T cells per antibody to be used at RT stimulated with 10 µM CHIR for 24 h at RT.
- Centrifuge cell solution 5 min at 600 x g at RT.
- Remove the liquid carefully.
- Gently resuspend cells in 1 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 20 µL NE Buffer per sample.
- Concanavalin A beads preparation
- Prepare one 2 mL microcentrifuge tube.
- Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6952467).
- Pipette 20 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 a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 20 µL per sample.
- Nuclei immobilization – binding to Concanavalin A beads
- Carefully vortex the nuclei suspension and add 20 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 200 µl of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) per sample.
- Primary antibody binding
- Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody.
- Add 2 µL antibody (anti-TCF7 antibody ABIN5620945, anti-H3K27me3 antibody positive control ABIN6923144, 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 100 µL of pAG mix per sample (100 µL of wash buffer + 58.5 µg 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 100 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
- Take the Mag-Bind® TotalPure NGS beads (Omega Bio-Tek, M1378-01) from the storage and wait until they are at 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 µL 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), 36 bp PE.
- Peak calling
- Trim reads using using bbTools bbduk (BBMap - Bushnell B. - sourceforge.net/projects/bbmap/) to remove adapters, artifacts and repeat sequences.
- Map aligned reads 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
Results are published in Zambanini, G. et al. A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets. bioRxiv (2022). https://doi.org/10.1101/2022.07.06.498999
生效 #104349 (Cleavage Under Targets and Release Using Nuclease)Validation ImagesFull Methods -
- 状态
- Liquid
- 缓冲液
- Supplied in liquid form in 0.42 % Potassium phosphate, 0.87 % Sodium chloride, pH 7.3 with 30 % glycerol and 0.01 % sodium azide
- 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
- 储存方法
- Store at 4 deg C for short term storage. For long term, aliquot and store at -20 deg C. Avoid repeat freeze/thaw cycles
-
-
A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets." in: Development (Cambridge, England), (2022) (PubMed).
: "
-
A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets." in: Development (Cambridge, England), (2022) (PubMed).
-
- 抗原
- TCF7 (Transcription Factor 7 (T-Cell Specific, HMG-Box) (TCF7))
- 别名
- TCF7 (TCF7 产品)
- 别名
- TCF-1 antibody, AI465550 antibody, Tcf1 antibody, Xtcf-1 antibody, Xtcf1 antibody, tcf-1 antibody, tcf1 antibody, tcf7 antibody, transcription factor 7 antibody, transcription factor 7, T cell specific antibody, transcription factor 7 S homeolog antibody, TCF7 antibody, Tcf7 antibody, tcf7 antibody, tcf7.S antibody
- 途径
- WNT signaling
-