Arginylation (N-Term) 抗体

ABIN4368250 产品详细信息, 供应商: Log in to see
抗原
抗原表位
N-Term
72
适用
化学剂
72
宿主

72
克隆类型
多克隆
标记
非结合性
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4
应用范围
Dot Blot (DB)
72
18
选项
Supplier
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Supplier Product No.
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'独立验证'标志
抗原 Arginylation (N-Term)
Method validated Western Blotting
Positive Control Arg-Asp-starting recombinant protein
Negative Control Asp-starting recombinant protein
Notes ABIN4368250 specifically detects recombinant protein with the N-terminal amino acid sequence Arg-Asp in a Western blot.
Primary Antibody ABIN4368250
Secondary Antibody goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004)
实验流程
  • Recombinant protein:
    • Bacterial protein expression and purification according to Naumann et al. 2016.
    • 1-15µg total protein were mixed with SDS-loading buffer and boiled for 5min.
  • Plant extracts:
    • Plant material (3 weeks old Arabidopsis) was ground with liquid nitrogen in RIPA-buffer and incubated for 15min at 4°C.
    • Debris was centrifuged at max. speed for 10min. Total protein content of the supernatant was determined.
    • 23.6µg (first experiment) or 94µg (second experiment) respectively were mixed with SDS-loading buffer and boiled for 5min.
  • Proteins were separated on a 12% denaturing SDS-PAGE gel (Carl Roth) in a Bio-Rad Tetra cell and transferred in a Bio-Rad semidry-blot apparatus for 1.5h at 1mA/cm2 to PVDF membrane (GE Healthcare).
  • The membrane was blocked in TBST with 5% milk powder (Carl Roth) for 1h in at RT.
  • Incubation with primary antibody ABIN4368250 (antibodies-online) diluted 1:1000 in TBST with 3% milk powder overnight at 4°C.
  • Washing with TBST with 3% milk powder for 3x 5min.
  • Incubation with secondary antibody goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) diluted 1:2500 in TBST with 3% milk powder for 1h at RT.
  • Washing with TBST with 3% milk powder for 5x 5min.
  • Detection with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) on a standard X-ray film; exposure 30s.
Experimental Notes ABIN4368250 recognizes the recombinant protein with N-terminal Arg-Asp shown in the image. In case of a different recombinant protein (not shown) the antibody did not only recognize the protein with the N-terminal sequence Arg-Asp but also with Asn or Asp at the amino-terminus.
Validation Images
Western Blotting validation image for anti-Arginylation (N-Term) antibody (ABIN4368250) Detection of detect a bacterially expressed recombinant protein with ABIN4368250, eit...
'独立验证'标志
抗原 Arginylation (N-Term)
Method validated Protein Array
Positive Control N-terminally arginylated synthetic peptides REHKHANQHMSVC, RDELPESIDWRKKGAV
Negative Control un-arginylated synthetic peptides HKHRDANQHMSVC, HKHREANQHMSVC, HKHANQHMSVA
Notes The antibody specifically recognizes peptides with the N-terminal amino acid sequence RD and to a lesser extent peptides with the N-terminal sequence RE.
Primary Antibody ABIN4368250
Secondary Antibody goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004)
实验流程
  • Peptide array synthesis according to Yim et al. 2015.
  • Block membrane with TBST with 7.5% milk powder overnight at 4°C.
  • Incubation with primary N-terminal arginylation antibody (antibodies-online, ABIN4368250) diluted 1:1000 in TBST with 3% milk powder for 1h at RT.
  • Washing with TBST for 3x 10min.
  • Transfer proteins onto PVDF membrane in a semi-dry for 30min at 0.8mA/cm2.
  • Block the membrane with TBST with 4% milk powder ON at 4°C.
  • Incubation with secondary antibody goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) diluted 1:2500 in TBST with 3% milk powder for 1h at RT.
  • Wash membrane 5x 20min in TBST.
  • Chemiluminescence detection with ECL (Thermo Scientific pico kit) on a standard X-ray film; exposure 30s.
Validation Images
Protein Array validation image for anti-Arginylation (N-Term) antibody (ABIN4368250) ABIN4368250 is highly specific for the sequence of the utilized antigen, i.e. N-termi...
免疫原 KLH-conjugated synthetic peptide: H-RDHKHANQHMSVC-NH2
特异性 N-terminal arginylation
纯化方法 Affinity purified serum. Antibodies are purified using substactive purification method.
物质类 Chemical
背景 Arginylation is a post-translational modification of an existing peptide chain by addition of an extra arginine. This modification changes primary sequence of protein as well as it's surface change. It is mediated by arginyltransferase ATE1. Arginylation plays an essential role in multiple physiological pathways, for example, in vivo arginylation constitutes a mechanism for degradation of preprocessed proteins or proteolytic fragments that bear Asp and Glu on their N-termini.
应用备注 1 : 1000 (Dot)
限制 仅限研究用
状态 Lyophilized
溶解方式 For reconstitution add 25 μL of sterile water to each tube.
缓冲液 PBS pH 7.4
储存条件 -20 °C
储存方法 Store lyophilized/reconstituted at -20°C, once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
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