* Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own experiment using appropriate negative and positive controls.
Antigen affinity chromatography
1mg/ml BSA, 30% glycerol
0.05% sodium azide
Product Specific Information
PA1118 has been used successfully in Western Blot to detect HRV3C cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro).
PA1118 has been used in Immunoprecipitation of an HRV3C cleavage site-containing protein from a whole Hela cell lysate.
Human rhinovirus 3C protease (HRV3C Protease) is a cysteine protease that recognizes the cleavage site of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro. It cleaves between Gln and Gly (independent of Pro). (HRV) 3C Protease is used to remove fusion tags from proteins with the HRV 3C cleavage sequence and is typically dual tagged for easy removal from the sample after cleavage.
HRV3C / Human rhinovirus 3C Antibody (PA1-118) in WB HRV3c_Antibody_PA1-118_Western-blot_1_20130919094950.jpg
Western Blot with anti-HRV3C / Human rhinovirus 3C Polyclonal Antibody (PA1-118)
Western blot analysis of HRV3c was performed by loading 1ug of a control protein, either uncleaved (lane 1) or cleaved (lane 2) using the Pierce Human Rhinovirus 3C Protease (HRV 3C) (Product # 88946) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an HRV3c polyclonal antibody (Product # PA1-118) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-rabbit IgG-HRP secondary antibody (Product # 31460) at a dilution of 1:15,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).
HRV3C / Human rhinovirus 3C Antibody (PA1-118) in IP HRV3c_Antibody_PA1-118_Immunoprecipitation_1_20130919094908.jpg
Immunoprecipitation with anti-HRV3C / Human rhinovirus 3C Polyclonal Antibody (PA1-118)
Immunoprecipitation of HRV3c was performed on a control protein containing GST and the HRV3c cleavage site. Antigen-antibody complexes were formed by incubating 100ug HeLa lysate containing 2ug control protein with 4ug of an HRV3c polyclonal antibody (Product # PA1-118) overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul of Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Both the lysate and the immune complexes were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with a GST monoclonal antibody (Product # MA4-004) at a dilution of 1:1500 overnight rotating at 4°C, washed in TBST, and probed with Clean-blot IP detection reagent (Product # 21230) at a dilution of 1:2500 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). Note: the IP fraction resulted in proteolytic activity of the GST-control protein.
(This product is for In Vitro experimental use only. Not for resale without express authorization.)