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CTNNB1 抗体 (Catenin (Cadherin-Associated Protein), beta 1, 88kDa) (Magnetic Particles)

Details for Product anti-CTNNB1 Antibody No. ABIN1690141, 供应商: Log in to see
抗原
  • Jup
  • LOC100217813
  • CTNNB1
  • CTNNB
  • Bfc
  • Catnb
  • Mesc
  • ctnnb
  • id:ibd2058
  • wu:fb73e10
  • wu:fi81c06
  • wu:fk25h01
  • ctnnb1
  • CHBCAT
  • MRD19
  • armadillo
  • beta-catenin
  • catenin (cadherin-associated protein), beta 1, 88kDa
  • catenin (cadherin associated protein), beta 1
  • catenin (cadherin-associated protein), beta 1
  • CTNNB1
  • LOC100217813
  • Ctnnb1
  • ctnnb1
  • ctnnb1-a
  • ctnnb1-b
Alternatives
anti-人 CTNNB1 抗体 for Immunofluorescence (fixed cells)
适用
人, 小鼠, 大鼠
473
212
188
62
53
42
26
24
22
20
15
15
15
10
9
9
3
1
1
1
1
1
1
宿主

308
179
5
2
克隆类型
多克隆
标记
This CTNNB1 antibody is conjugated to Magnetic Particles
14
11
8
7
6
6
4
3
2
2
1
1
1
1
1
1
1
应用范围
Immunoprecipitation (IP)
437
150
127
125
108
96
70
31
16
13
8
8
4
3
3
2
1
1
Supplier
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Supplier Product No.
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Available images

'独立验证'标志
抗原 CTNNB1
Lot Number 416
Method validated Immunoprecipitation
Positive Control Colo205 human colorectal cell line
Notes ABIN1690141 (CTNNB1 antibody conjugated to magnetic particles) was found to be suitable for IP of beta-catenin from lysates of Colo205 cells.
Primary Antibody ABIN1690141
实验流程
  • Cell lysates:
    • Prepare detergent-free lysates of Colo205 cells on ice in low detergent 'soft' elution buffer (150mM NaCl, 50mM Tris-HCl, 0.02% Tween-20, pH8.0; see Antrobus R and Borner GH, (2011) PLoS One) supplemented with Halt Protease and Phosphatase inhibitor cocktail (Thermo Fisher, 78440, lot RE234421) using a TissueRuptor (Qiagen).
    • Pellet insoluble debris by centrifugation.
    • Transfer the supernatant and quantitate the total protein concentration by CB-X Protein Assay (G Biosciences, 786-11X, lot 152710).
  • Immunoprecipitation:
    • Dilute supernatant to 1mg/ml total protein.
    • Equilibrate CTNNB1 antibody magnetic particles (antibodies-online, ABIN1690141, lot 0416) with PBS and then wash them 3x with PBS to remove residual detergent.
    • Incubate 50µg CTNNB1 antibody magnetic particles (antibodies-online, ABIN1690141, lot 0416) with 1ml of Colo205 lysate at 1mg/ml total protein ON at 4°C on an end-over-end rotator.
    • Recover the magnetic beads on a Dynabead rack (ThermoFisher). Store the removed supernatant at -80°C for the subsequent immunoblot.
    • Wash beads 5x with 1ml ice cold PBS supplemented with Halt cocktail. Store each wash fraction at -80°C for the subsequent immunoblot.
    • Incubate immunoprecipitated proteins with 50µl 'soft' reduced detergent elution buffer (0.2% (w/v) SDS, 0.1% (v/v) Tween-20, 50mM Tris-HCl pH8.0) suitable for downstream mass spectrometric analysis.
  • Immunoblot:
    • Separate proteins on a 10% SDS-PAGE gel using a Mini-PROTEAN 3 cell electrophoresis tank (Bio-Rad, 165-3301/165-3302) at 200 V for 90min at RT and Tris-Glycine-SDS running buffer.
    • Transfer proteins to PVDF immobilon-P membrane (Millipore, IPVH00010, R6kA7917F) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, 170-3930/170-3935) at 30V, 90mA, ON at 4°C in Tris-Glycine transfer buffer.
    • Incubate with a primary mouse purified mouse-anti-beta-catenin antibody (BD Biosciences, 610154, LOT 5113978) diluted 1:1000x in TBST (TBS, 0.1% (v/v) Tween-20) with 5% non-fat milk for 2h at RT.
    • Wash membrane 3x 5min with TBST.
    • Incubate with HRP-conjugated polyclonal goat anti-mouse secondary antibody (Dako, P0447, lot 00082889) diluted 1:2000 in TBST with 5% non-fat milk for 1h at RT.
    • Wash membrane 3x 5min with TBST.
    • Visualized immunoreactive bands by autoradiography. Incubate membrane for 1min at RT with 3ml of ECL Western Blotting Substrate (Promega, W1001, lot 0000193677) and expose to Hyperfilm ECL (Amersham, product 28906837, lot 64701) for 5min in the dark.
  • Mass spectrometric analysis:
    • Extract peptides from the gel band and submit to a tryptic digest with Sequencing Grade Modified Trypsin (Promega, V5111).
    • Run tryptic fragments on a Q Exactive (QEX) Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher).
    • Identify proteins fragments by performing an MS/MS Ion search with MASCOT (Matrix Science) against the UPR HomoSapiens 20160706 database (92,578 sequences; 36,833,215 residues).
Experimental Notes
  • ABIN1690141 proved to be be susceptible to detergents as it was evidenced by the higher efficacy of the lower detergent 'soft' elution buffer compared to RIPA buffer with PBS.
  • The IP can be improved through the use of detergent-free protein lysate preparations and by washing with PBS rather than RIPA, as strong detergents can strip beta-catenin from the antibodies on the beads. The 'soft', low detergent elution buffer formulated by Antrobus and Borner is recommended where the eluted protein is to be used for mass spectrometry.
  • Assuming that the lysate for the IP came from the same source, so there is no danger of cross-contamination, the beads can be re-used approximately four times.
Validation Images
Immunoprecipitation validation image for anti-Catenin (Cadherin-Associated Protein), beta 1, 88kDa (CTNNB1) antibody (Magnetic Particles) (ABIN1690141) A. SDS-PAGE of protein fractions from immunoprecipitation of beta-catenin with ABIN16...
别名 CTNNB1 / Beta Catenin (CTNNB1 Antibody 摘要)
途径 WNT signaling, Intracellular Steroid Hormone Receptor Signaling Pathway, Peptide Hormone Metabolism, Regulation of Muscle Cell Differentiation, Cell-Cell Junction Organization, Tube Formation, Maintenance of Protein Location, Signaling Events mediated by VEGFR1 and VEGFR2
限制 仅限研究用