Ni-NTA HisSorb Strips (24)

Protocol tips

Upstream tips	Protocol tips	Downstream tips

Publication protocol

Enzyme-linked Immunosorbent Assays: Wells of Ni-NTA HisSorb strips (Qiagen) were coated with 100 μl of His-tagged integrin model proteins or His-tagged FAT, a recombinant protein derived from the focal adhesion targeting sequence of focal adhesion kinase (FAK), dissolved in phosphate-buffered saline (PBS) plus 0.2% BSA at 4 °C overnight. The next day, wells were washed with PBS three times and blocked with 150 μl of 1% (w/v) heat-denatured BSA at room temperature for 1 h. The wells were then washed with PBS three times. 100 μl of recombinant paxillin or its mutants at different concentrations dissolved in PBS plus 0.2% (w/v) BSA was added to each well and incubated at room temperature for 1 h. Unbound proteins were washed out with PBS three times. Bound proteins were stained with mouse anti-HA tag (1:1,000 dilution in PBS plus 1% BSA) or mouse anti-GST antibodies (1:1,000 dilution in PBS plus 1% BSA) for 1 h at room temperature, followed by 1 h incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (1:1,000 dilution in PBS plus 1% BSA) (BioSource). After three washes, bound proteins were assayed by measuring peroxidase activity with o- phenylenediamine as a substrate and quantified by reading its optical density at 490 nm. For competition assays using paxillin fragment, P(Ala176–Asp275), or recombinant full-length paxillin (GST-free), the same modified ELISA assays were performed except that different concentrations of this fragment was included in the paxillin solution added to the integrin model protein- or FAT-coated wells. Data were expressed as percentage of inhibition: (1 − B/B0) × 100%; where B = A490 in the presence of the competitor and B0 = A490in its absence.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein tag Detection of His-tagged proteins using Ni-NTA HisSorb Strips (24) from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for Ni-NTA HisSorb Strips (24) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Protein tag Detection of His-tagged proteins using Ni-NTA HisSorb Strips (24) from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.