RGS·His HRP Conjugate Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Follow manufacturer’s instructions

Protocol tips
Follow manufacturer’s instructions

Publication protocol

Heterologous Expression and Purification of P. brasiliensis Agn1p: For Agn1p-his purification, the P. brasiliensis AGN1 ORF (Gen-Bank Accession No. EF679780) deprived of the sequence coding for the putative signal peptide was PCR-amplified from cDNA, using primers HVC2 5′-CAT AGA GCT CAT TCA AAC ATC CAC GCT-3′ and HVC3 (5′-GGA TCC AAG GCT GTA TTT GCC CAT TTC-3′), and cloned at the SacI and BamHI restriction sites of plasmid pQE30Xa (Qiagen), generating pHV2. E.coli M15 [pREP4] (Qiagen, Hilden, Germany) was transformed with pHV2 or pQE30Xa empty (as negative control), grown on LB medium supplemented with 100 µg/ml ampicillin (Sigma-Aldrich, St Louis, MO, EE.UU) and 25 µg/ml kanamycin (Sigma-Aldrich, St Louis, MO, EE.UU) at 37°C, following the manufacturer’s indications. For protein expression experiments, each transformant was grown on LB medium supplemented with 100 µg/ml ampicillin (Sigma-Aldrich, St Louis, MO, EE.UU), 25 µg/ml kanamycin (Sigma-Aldrich, St Louis, MO, EE.UU), 500 mM NaCl, 0.2% glucose and 1 mM sorbitol at 37°C for 5h until culture OD600 reached 0.7. Protein expression was induced with the addition of 0.5 mM IPTG and cultures were grown at 23°C overnight [15], [32]. Cells were harvested by centrifugation (20 min, 4000g, 4°C) and washed with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Cell pellets were resuspended in lysis buffer supplemented with 1 µl/ml protease inhibitor cocktail (Sigma P-8215), treated with 1 µg/ml lysozyme (Sigma L-6876) on ice for 30 min. Cells were lysed with 0.17 µm glass beads [33], in a Braun homogenizer (Braun, Melsungen, Germany), using 4 pulses of 1 min each, with 1 min cooling on ice between pulses. Cell debris was removed by centrifugation at 4°C at 10000×g for 15 min. Clear lysates were incubated with pre-washed nickel-NTA resin (QIAGEN) at 4°C for 1 h, and subsequently washed with buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Agn1p-his was eluted in fractions by addition of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluates were pooled and concentrated to 1 ml using Amicon ® Ultra-4 (Millipore) with a 30 kDa cutoff. The concentration process was performed at 4000g for 90 min at 4°C. Purity was monitored by SDS-PAGE analysis employing Mini-PROTEAN chambers ® II Electrophoresis Cell (Bio-Rad, Hercules, CA, USA), as recommended by the manufacturer and according to the size of the expected product [34], [35]. Separation and stacking gels of 10 or 4% polyacrylamide were used. The following molecular weight standards were employed: Prestained marker (98,5 - 14) kDa (26041-020, Gibco-BRL) and 6xHis Protein Ladder (100-15) kDa (34705, QIAGEN). Immunodetection of the purified protein was performed with the chromogenic method described in the QIAexpress ® Detection System manual (Qiagen, Hilden, Germany), using the HRP Conjugate Kit RGS-His.

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 RGS·His HRP Conjugate Kit from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for RGS·His HRP Conjugate Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Protein tag Detection of His-tagged proteins using RGS·His HRP Conjugate Kit from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.