Anti-Human SUMO1 Affimer Reagent (HS1-A1) Biotinylated

Catalogue CodeAVA00024-Biotin Size100μg
 
Affimer® (HS1-A1) to Human SUMO1 Biotinylated

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Anti-Human SUMO1 Affimer Reagent (HS1-A1) Biotinylated
£200.00
 
Catalogue CodeAVA00024-Biotin Size100μg
 
Affimer® (HS1-A1) to Human SUMO1 Biotinylated
Conjugate
OR
Datasheet

Datasheet

General  
Name Anti-Human SUMO1 Affimer Reagent (HS1-A1) Biotinylated
Catalogue Code AVA00024-Biotin
Overview  
Clone ID HS1-A1
Size (μg) 100
Tested Applications Co-Immunoprecipitation, ITC, Western Blotting
Tags C-term 6His
Conjugate Biotinylated
Properties  
Form Liquid
Storage Instructions For short term use, store at 4°C. We recommend aliquoting and storing at -20°C long term. Affimers are generally unaffected by 3-4 freeze/thaw cycles
Buffer 100mM Sodium Phosphate, 75mM Sodium Chloride, 0.02% Sodium Azide, pH 7.4
Purity >95%
Purification Method IMAC followed by desalting
Clonality Monoclonal
Target  
Target Human SUMO1
Species Reactivity Human
Target Uniprot ID P63165
Target Function

Ubiquitin-like protein that can be covalently attached to proteins as a monomer or a lysine-linked polymer. Covalent attachment via an isopeptide bond to its substrates requires prior activation by the E1 complex SAE1-SAE2 and linkage to the E2 enzyme UBE2I, and can be promoted by E3 ligases such as PIAS1-4, RANBP2 or CBX4. This post-translational modification on lysine residues of proteins plays a crucial role in a number of cellular processes such as nuclear transport, DNA replication and repair, mitosis and signal transduction. Involved for instance in targeting RANGAP1 to the nuclear pore complex protein RANBP2. Polymeric SUMO1 chains are also susceptible to polyubiquitination which functions as a signal for proteasomal degradation of modified proteins. May also regulate a network of genes involved in palate development.

Affimers have been designed to perform better than antibodies in a number of areas.

Research Area Cell Signalling, UPS
Applications

Applications

This Affimer has only been tested in the applications detailed below. The application notes include recommended starting concentrations, however, it is expected that the optimal conditions be determined by the end user.

Isothermal Titration Calorimetry

AVA00024 Isothermal Titration Calorimetry

Clone HS1-A1 was shown to specific for Human SUMO1 (Kd = 97nM) but has no interaction with Human SUMO2 by isothermal titration calorimetry.

Co-Immunoprecipitation

AVA00024 Co-Immunoprecipitation

HEK293T cells were (co)-transfected with plasmids bearing the target (GFP, GFP-SUMO1 or GFP-SUMO2) and Affimer (with FLAG tags) for 48 h. Cells were washed in PBS and proteins extracted in 1 ml lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40 and 1x protease inhibitor cocktail (Roche) for 15 min on ice and clarified by centrifugation at 12,000 xg for 10 min, 4°C. Lysates were incubated with GFP-Trap (Chromotek) for co-immunoprecipitation of GFP-labelled species. Immunoprecipitated proteins were eluted in Laemmli buffer and subject to immunoblot analysis. As marked but the red box, Affimer (HS1-A1) co-precipitates in the presence of GFP-SUMO1 but not GFP-SUMO2.

Western Blotting

AVA00024 Western Blotting
SDS-PAGE & Blotting
Protein samples (100 ng per well) were separated on a Bolt 4-12% Bis-Tris Plus gel (Life Tech) and blotted onto Nitrocellulose using an iBlot2 (Life Tech).
Blocking
The membrane was blocked using 1 x TBS + 3% skimmed milk powder (1 h, RT°C, 400 rpm)
Wash 1
3 x 5ml of 1xTBS-T (0.05% Tween-20)
Affimer Incubation
5μg/ml in 1 x TBS + 3% skimmed milk powder (16 h, 4°C, 400 rpm)
Wash 2
3 x 5ml of 1xTBS-T (0.05% Tween-20)
Detection
Rabbit anti-6xHis HRP conjugate, 1/5,000 dilution in 1 x TBS + 3% skimmed milk powder (1 h, RT°C, 400 rpm)
Wash 3
3 x 5ml of 1xTBS-T (0.05% Tween-20)
Substrate
ECL (Amersham)

A detectable complex is only formed when the Affimer is present: The Affimer recognises the immunoglobulin and detection of the complex is mediated via the 6xHis tag on the Affimer. The anti-6xHis binder does not detect the IgG on membrane 4 where the Affimer is absent.

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