INPP5E 抗体 (C-Term)
(1 validation)Quick Overview for INPP5E 抗体 (C-Term) (ABIN7298962)
抗原
See all INPP5E 抗体适用
宿主
克隆类型
标记
应用范围
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抗原表位
- C-Term
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特异性
- Recognizes endogenous levels of INPP5E protein.
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产品特性
- Rabbit polyclonal antibody to INPP5E
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纯化方法
- The antibody was purified by immunogen affinity chromatography.
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免疫原
- KLH-conjugated synthetic peptide encompassing a sequence within the C-term region of human INPP5E.
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anti-Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E) (AA 1-133) antibody
INPP5E 适用: 人 ELISA, IHC, IF 宿主: 兔 Polyclonal unconjugated
anti-Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E) (AA 510-630) antibodyINPP5E 适用: 人 WB, IF 宿主: 兔 Polyclonal unconjugated
anti-Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E) (AA 1-644) antibodyINPP5E 适用: 人 WB 宿主: 兔 Polyclonal unconjugated
anti-Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E) (AA 1-644) antibodyINPP5E 适用: 人 WB 宿主: 小鼠 Polyclonal unconjugated
anti-Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E) (C-Term) antibodyINPP5E 适用: 人, 小鼠, 大鼠 WB, ELISA 宿主: 兔 Polyclonal unconjugated
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应用备注
- WB (1:500 - 1:1000)
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限制
- 仅限研究用
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- by
- Children's Hospital of Eastern Ontario Research Institute
- No.
- #101129
- 日期
- 2017.11.10
- 抗原
- INPP5E
- Lot Number
- CP1D10A
- Method validated
- Western Blotting
- Positive Control
- Inpp5e+/+ 4T1 mouse breast carcinoma cell line (transfected with non-targeting Cas9 gRNA), treated with an inducer (unpublished)
- Negative Control
- Inpp5eem 4T1 cell line (CRISPR/Cas9 endonuclease mediated Inpp5e knock-out)
- Notes
Passed. ABIN2704925 specifically recognizes Inpp5e in murine Inpp5e+/+ 4T1 cells upon induction aside from other unspecific bands.
- Primary Antibody
- ABIN2704925
- Secondary Antibody
- goat anti-rabbit IgG (H+L)-HRP-conjugated antibody (BioRad, 172-1019, lot 350001898)
- Full Protocol
- Grow Inpp5e+/+ and Inpp5eem 4T1 murine cells in HyClone TM RPMI-1640 medium (GE Healthcare Life Science, SH30027.01, lot AC11016269) supplemented with fetal bovine serum (Sigma, F1051-500ML, lot 17D192) and HyClone penicillin – streptomycine solution (GE Healthcare Life Science, SV30010, lot SH40003.01), at 37°C and 5% CO2 in 3ml on a 6-well plate to 95% confluency.
- Treat cells with an “inducer” (unpublished) to increase Inpp5e expression.
- Lyse cells in 200µl per well of cold RIPA buffer (150mM NaCl, 1.0% IGEPAL CA-630 Sigma, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris, pH8.0).
- Determine the total protein content of the lysates using DC Protein Assay (BioRad, 500-0114, lot 200008008).
- Denature 30µg of total protein for 5min at 95°C in 5x Laemmli SDS sample buffer and subsequently separate proteins on a non-/denaturing 10% SDS-polyacrylamide gel in a mini-Protean electrophoresis cell (BioRad) for 90min at 100V.
- Transfer proteins onto 0.2µm nitrocellulose membrane (BioRad, 1620112, lot A10088580) with a MiniTransblot Cell (BioRad, 1703930) system for 60V at 300mA.
- Block the membrane with TBS containing 0.1% Tween 20 (TBST) and 5% skim milk for 30min at RT.
- Incubation with primary rabbit anti-INNP5E antibody (antibodies-online, ABIN2704925, CP1D10A) diluted 1:1000 in TBST containing 5% BSA and 0.05% NaN3 ON at 4°C.
- Wash membrane 3x with TBST.
- Incubation with secondary goat anti-rabbit IgG (H+L)-HRP-conjugated antibody (BioRad, 172-1019, lot 350001898) diluted 1:5000 in TBST for 1h at RT.
- Wash membrane 5x with TBST.
- Reveal protein bands using Clarity Western ECL Substrate (BioRad, 1705060, lot 102030837) on a ChemiDoc Imaging System (Biorad, 17001401).
- Experimental Notes
ABIN2704925 reveals a single band in the "wild type" cell line with treatment. This protein band is abrogated in the CRISPR/Cas9 targeted cells and under non-inducing conditions, thus demonstrating immunospecificity of ABIN2704925 for Inpp5e in the mouse.
We observed a lower molecular weight than the predicted size of 72kDa for Inpp5e. We suspect that this might be a splice isoform. We observed a similar band also with a different anti-INPP5E antibody (Thermo Fisher Scientific, PA5-37119) using similar experiment condition.
- ABIN2704925 also recognizes potentially unspecific bands at various sizes.
生效 #101129 (Western Blotting)!['独立验证'标志]( "成功验证")
!['独立验证'标志]( "成功验证")
Validation Images![Western blot of 4T1 mouse breast carcinoma cell line extracts (Inpp5e+/+, lane 1 and 2) and 4T1 lnpp5e CRISPR/Cas9 knock-out cell extracts (Inpp5eem, lane 3 and 4) with ABIN2704925. A single band appears upon cell treatment with an inducer (unpublished) specifically in the Inpp5e+/+ cells.]()
Western blot of 4T1 mouse breast carcinoma cell line extracts (Inpp5e+/+, lane 1 and 2) and 4T1 lnpp5e CRISPR/Cas9 knock-out cell extracts (Inpp5eem, lane 3 and 4) with ABIN2704925. A single band appears upon cell treatment with an inducer (unpublished) specifically in the Inpp5e+/+ cells.
Full Methods -
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状态
- Liquid
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缓冲液
- Liquid in 0.42 % Potassium phosphate, 0.87 % Sodium chloride, pH 7.3, 30 % glycerol, and 0.01 % sodium azide.
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储存液
- Sodium azide
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注意事项
- This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
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储存条件
- -20 °C
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储存方法
- Shipped at 4°C. Upon delivery aliquot and store at -20°C for one year. Avoid freeze/thaw cycles.
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有效期
- 12 months
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