STAT5A 抗体
Quick Overview for STAT5A 抗体 (ABIN5557532)
抗原
See all STAT5A 抗体适用
宿主
克隆类型
标记
应用范围
克隆位点
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                                            交叉反应
- 人, 小鼠, 大鼠
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                                            纯化方法
- Purified by Protein A.
- 
                                            免疫原
- Recombinant human STAT5a protein, around C-terminal 100aa.
- 
                                            亚型
- IgG
 
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                                            应用备注
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                        WB 1:300-5000
 IHC-P 1:200-400
 IF(ICC) 1:50-200
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                                            限制
- 仅限研究用
 
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- 
        - by
- Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
- No.
- #104384
- 日期
- 2022.12.07
- 抗原
- STAT5A
- Lot Number
- BA7271967
- Method validated
- Cleavage Under Targets and Release Using Nuclease
- Positive Control
- Polyclonal rabbit anti-H3K4me (antibodies-online, ABIN3023251) 
- Negative Control
- Polyclonal guinea pig anti-rabbit IgG (antibodies-online, ABIN101961) 
- Notes
- Passed. ABIN5557532 allows for CUT&RUN targeting of STAT5A in mouse forelimb tissue. 
- Primary Antibody
- ABIN5557532
- Secondary Antibody
- Full Protocol
- Cell harvest and nuclear extraction
- Dissect 3 Fore limbs (11.5 DAC) from mouse strain RjOrl:SWISS for each sample.
- Dissociate the tissue into single cells in TrypLE (Thermo Fisher Scientific) for 15 min at 37 °C.
- Centrifuge cell solution 5 min at 800 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 (150,000 cells per sample).
- Add 2 µL antibody (anti-STAT5A antibody ABIN5557532, anti-H3K4me positive control antibody 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 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-HCl pH 8.2.
- 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-HCl pH 8.2.
 
- 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
- The protocol is published in Zambanini, G. et al. A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets. Development (2022). PMID 36355069 
 生效 #104384 (Cleavage Under Targets and Release Using Nuclease)![成功验证 '独立验证'标志]()  ![成功验证 '独立验证'标志]() Validation ImagesFull Methods Validation ImagesFull Methods
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                                            状态
- Liquid
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                                            浓度
- 1 μg/μL
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                                            缓冲液
- Aqueous buffered solution containing 1xTBS ( pH 7.4), 1 % BSA, 40 %Glycerol and 0.05 % Sodium Azide.
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                                            储存液
- ProClin
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                                            注意事项
- This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.
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                                            储存条件
- 4 °C,-20 °C
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                                            储存方法
- Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.
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                                            有效期
- 12 months
 
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    - STAT5A (Signal Transducer and Activator of Transcription 5A (STAT5A))
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                                            别名
- STAT5a
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                                            背景
- 
                        Synonyms: MGF, STAT5, Signal transducer and activator of transcription 5A, STAT5A Background: Carries out a dual function: signal transduction and activation of transcription. Mediates cellular responses to the cytokine KITLG/SCF and other growth factors. Mediates cellular responses to ERBB4. May mediate cellular responses to activated FGFR1, FGFR2, FGFR3 and FGFR4. Binds to the GAS element and activates PRL-induced transcription. Regulates the expression of milk proteins during lactation. 
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                                            基因ID
- 6776
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                                            UniProt
- P42229
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                                            途径
- JAK/STAT Signaling, RTK signaling, Response to Growth Hormone Stimulus, C21-Steroid Hormone Metabolic Process, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, CXCR4-mediated Signaling Events, Activated T Cell Proliferation
 抗原
- 
                    
 
                                                 
                                                 (1 validation)
 (1 validation) 
                 
                                     
                                     
                                    