Protein expression and anti-protein antibodies

From your vector to purified Ab

Reference: IAB09-03
From vector to purified Ab

Type: Affinity purified

Host: 2 rabbits

Material provided by the customer:

Complete expression plasmid vector (5ug)
Complete sequence
Restriction map

Service features:

Protein expression and purification ;
4 immunisations (rabbit), 70 days ;

Affinity chromatography on protein column

ELISA, guaranteed titer >1:30 000
Western blot (WB) provided

Delivery

2-5 mg specific antibodies
Pre-immune sera
Report with ELISA data
Western Blot against expressed protein
0.5-1 mg of protein (purity >90%)

Leadtime

4 month

Price :
2.475 €

WB protocol - General Guidelines

>0.200 – 0.025 µg of total protein from a pure stock of protein in 1x SIGMAFAST EDTA-free Protease Inhibitor (S8830-2TAB) and denatured with 1x reducing Laemmli SDS buffer at 90°C for 10 min, and insoluble material pelleted at 20,000 xg for 15 min.
Samples were run to separation on a 4-12% SDS-PAGE gel and blotted 7 mins to PVDF using ThermoFisher iBlot high voltage Protocol 3. Blot was blocked with 7.5 % milk in TBS-T for 20mins/RT with agitation.

Blot was incubated in the primary antibody (i.e. Rabbit anti-protein-IgG) at a dilution of 1:2.000 with 7.5 % milk in TBS-T OverNight/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly three times, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation.
Blot was blocked with 7.5 % milk in TBS-T for 20mins/RT. Blot was then incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in TBS-T for 2.5h/RT with agitation. The blot was washed as above with TBS, and developed for 5 min with chemiluminescent substrate.

MW markers: BenchMark™ Pre-stained Protein Ladder (10748010) (5)

Zugriffe heute: 3 - gesamt: 4160.