Cultures were grown as described in sample preparation for live microscopy. Aliquots of 1 ml were centrifuged for 3 min at 10,000 × g and resuspended in 30 µl of Laemmli sample buffer and denatured for 15 min to lyse the cells. Samples were run on a 15% SDS-PAGE gel (30 mA, 30 min), blotted on Sequi-Blot PVDF membrane (Bio-Rad #1620182) and blocked with 8% Marvel dried skimmed milk in Tris-buffered saline buffer with Tween 20 (TBST buffer) overnight at room temperature. Blots were probed with primary rabbit anti-Pal (1:1000) and anti-TolB (1:500) antibodies in 4% milk in TBST buffer for 1 h at room temperature. Membranes were then washed with TBST buffer (5 × 1 min) and probed with secondary goat anti-rabbit antibodies conjugated with peroxidase (1:1000, Sigma #A6154). Blots were washed as described above and detection was carried out using Amersham ECL Western Blotting Select Detection Reagent (GE Lifesciences #RPN2235), according to the manufacturer’s instructions in GBOX-CHEMI-XRQ. Images were recorded using GeneSys software.
Goat anti rabbit antibodies conjugated with peroxidase
Manufactured by Merck Group
Goat anti-rabbit antibodies conjugated with peroxidase are secondary antibodies used in various immunoassay techniques. They are designed to specifically bind to primary rabbit antibodies, enabling detection and amplification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using goat anti rabbit antibodies conjugated with peroxidase
1
Western Blot Analysis of Bacterial Proteins
2
Quantification of Anti-EGF VacAbs in NSCLC Patients
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Sera from patients enrolled in the BV-NSCLC-001 clinical trial (NCT01444118) were kindly provided by InBio Europe Ltd. BV-NSCLC-001 was a randomized trial to study the safety and efficacy of anti-EGF VacAbs in late-stage (IIIB/IV) NSCLC patients, who were immunized with a first generation anti-EGF vaccine [30 (link),31 (link)]. All patients provided written and informed consent. The concentration of hEGF in sera was assayed using the Quantiquine ELISA hEGF immunoassay (R&D systems, Minneapolis, MN), according to the manufacturer's instructions. The titer of anti-EGF VacAbs in rabbit and patient's sera were determined using an in-house ELISA. Briefly, recombinant hEGF (2 μg/mL) (Sigma-Aldrich) was attached to the wells of a flat bottom 96-wells plate, wells were blocked, incubated with serial dilutions of patient's sera followed by goat anti-rabbit antibodies conjugated with peroxidase (Sigma- Aldrich). In head-to-head comparisons of anti-EGF VacAbs with cetuximab and panitumumab, the same protein concentrations were used. Finally, a substrate solution was added for 20 min, the reaction stopped with 1 N NaOH (Macron Fine Chemicals, Radnor, PA) and plates read at 405 nm with Infinite M Plex microplate reader.
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