MA1-086

antibody from Invitrogen Antibodies

Targeting: ATIC AICARFT, IMPCHASE, PURH

Provider product page for MA1-086

Western blot

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [1]
Comments [0]
Validations
Western blot [2]
Immunohistochemistry [1]
Other assay [2]

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Product number: MA1-086 - Provider product page
Provider: Invitrogen Antibodies
Product name: ATIC Monoclonal Antibody (F38 P7 H9)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA1-086 detects the ATIC in human, rat, Xenopus, Drosophila, and mouse cells.
Antibody clone number: F38 P7 H9
Concentration: 1 mg/mL

ATIC protein structure - MA1-086 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extract (30 µg) of Hep G2 (Lane 1) HCT 116 (Lane 2) K-562 (Lane 3), COLO 205 (Lane 4), NTERA-2 (Lane 5) and HT -29 (Lane 6). The blots were probed with Anti-ATIC Mouse Monoclonal Antibody (Product # MA1-086, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~ 65 kDa band corresponding to ATIC was observed across cell lines. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ATIC was achieved by transfecting HEK 293 cells with ATIC specific siRNAs (Silencer® select Product # s1706). Western blot analysis (Fig a) was performed using whole cell extracts from the ATIC knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti- ATIC Mouse monoclonal Antibody (Product # MA1-086, 2 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to ATIC.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Tumor-associated autoantibody XC154mAb was identified in human (HCC) model HBx-Tg mouse. ( A ) SDS-PAGE analysis of purified XC154 mAb. Purified XC154 mAb (10 mug) was treated with non-reducing (NR) or reducing (R) SDS-PAGE sample buffer and separated on 10% SDS-PAGE gel. Coomassie blue stained gel showed high molecular weight IgM and mu heavy chain with molecular weight of 72 kDa. M: molecular weight marker. ( B ) The expression of XC154 Ag in liver tissues of H- ras 12V-Tg mice. The liver tissue lysates (50 mug) of wild-type mice ( n = 3) or tumor-bearing H- ras 12V-Tg mice ( n = 6) were separated on 10% SDS-PAGE and Western Blots were probed with XC154 mAb. Band intensities were quantified by Image J software and the values were normalized to beta-actin. ( C ) Expression of XC154 antigen in various human tumor cell lines (cell lysates 40 mug) shown by Western blotting. GAPDH was served as an internal control. Arrows indicate the XC154 antigen. ( D ) Immunofluorescent staining of tumor cell lines with XC154 mAb (0.5 mug/mL) and FITC-labeled anti-mouse IgG. ( E ) Immunohistochemical staining of human liver tissues (non-neoplatic or HCC tissue) microarray with XC154 mAb (0.5 mug/mL). DAB intensities were quantified by Image J software and the relative values were plotted. Statistical significance was determined by two-tailed Student's t -test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 The target antigen of XC154 mAb was oncogenic ATIC. ( A ) Preparative 10% SDS-PAGE to isolate XC154 antigen and in-gel digestion for the mass spectrometric protein identification. The protein band containing XC154 antigen, which was confirmed by Western blotting, was excised (indicated by the red arrow) and in-gel digested. Proteins identified by mass spectrometric analysis were listed in Table 1 . ( B ) The validation of XC154 antigen as ATIC. HepG2 cells were transfected with si-ATIC and their cell lysates were analyzed by Western blotting with XC154 mAb. beta-Actin was used as an internal control. ( C ) Immunoprecipitation analysis for the verification of XC154 antigen as ATIC. Red arrows indicate XC154 Ag or ATIC. ( D ) The expression of ATIC in liver tissues of H- ras12V -Tg mice. Blots were probed with commercial anti-ATIC antibody. ( E ) Immunohistochemical analysis of ATIC in liver tissues of HCC model mice. Liver tissues from wild type control mice (Non-Tg: WT) were also stained. NT: non-tumor, T: tumor region, H- ras12V -Tg ( n = 6), Non-Tg ( n = 3), HBx-Tg-nonT: HBx-transgenic mouse without tumor ( n = 5), HBx-Tg-ST: HBx-transgenic mouse with small tumor ( n = 4), HBx-Tg-LT: HBx-transgenic mouse with large tumor ( n = 7). Representative images were shown (All staining images were shown in Supplementary Figure S2 ). ( F ) ATIC in exosomes purified from hepatoma cell cultured media (HepG2 and Huh7 cells; 35 mug) analyzed by Western blotting. A well-known exo