The pcDNA3.3-TOPO vectors, containing the coding sequences for the RECK gene variants, plus a C-terminal His-Tag, were transfected into 293T cells using the Lipofectamine 2000 reagent (Life Technologies), according to the manufacturers instructions, and, after 4 days of culturing, the cell culture medium was collected and the cellular extracts obtained by lysing the cells with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% SDS, 50 mM Tris pH8, 2 mM EDTA). 100 μL of these conditioned media were resolved on a 12.5% SDS-Page gel and transferred to a nitrocellulose membrane. The membranes were blocked for one hour in PBS-0.1% Tween 20 and 5% milk, followed by incubation for 1 h at room temperature with the Anti-His-Tag antibody (GE Helathcare, Buckinghamshire, UK) diluted 1:1,000 in PBS-0.1% Tween 20 (PBST) and 5% milk. After three washes of 10 min each with PBST, the membranes were incubated with the anti-mouse antibody (Qiagen) diluted 1:2,000 in PBST and 5% milk for one hour. Signal was detected the using Immobilon Western Chemiluminescent HPR substrate (Merck Millipore, Darmstadt, Germany).
Anti his tag antibody
Manufactured by GE Healthcare
Sourced in United Kingdom, Germany
The Anti-His tag antibody is a laboratory tool used to detect and purify proteins that have been engineered to contain a histidine (His) tag. The antibody specifically binds to the His tag, allowing the tagged protein to be identified and isolated from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemiluminescent hpr substrate, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Hybond p, by GE Healthcare (1 mentions)
Las 1000 uv mini, by Fujifilm (1 mentions)
Immobilon western chemiluminescent substrate, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by GE Healthcare (1 mentions)
Cm12 microscope, by Philips (1 mentions)
4 protocols using anti his tag antibody
1
RECK Variant Quantification in 293T Cells
2
Western Blot Analysis of AS3MT Protein
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HepG2 cells treated with each siRNA were washed with ice-cold PBS(−) and collected with 50 mM Tris–HCl (pH 7.4). The pellets were homogenized by sonication for 15 min, and then centrifuged at 105,000 × g for 1 h. A 20 μg portion of protein in the supernatant was separated by SDS-PAGE, and then transferred onto polyvinylidene fluoride membrane (Hybond-P, GE Healthcare) at 20 V for 1 h. The membrane was blocked for 1 h with 3% BSA in PBS(−) containing 0.1% Tween-20 (PBS-T). For the detection of AS3MT, the membrane was washed briefly with PBS-T and incubated with anti-Cyt19 (AS3MT) rabbit polyclonal antibody (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 100-fold with PBS-T containing 5% BSA overnight at 4 °C. The membrane was washed with PBS-T, and probed with horseradish-peroxidase-conjugated anti-rabbit IgG (1:50,000) (GE Healthcare). The bands were visualized with Immobilon Western Chemiluminescent Substrate (Merck Millipore, Billerica, MA, USA) and LAS-1000 UV mini (FUJIFILM, Tokyo, Japan). For the detection of rhAS3MT, anti-His-tag antibody (GE Healthcare) was also used as the primary antibody in addition to anti-Cyt19 antibody. Anti-GAPDH mouse polyclonal antibody (Santa Cruz Biotechnology) and horseradish-peroxidase-conjugated anti-mouse IgG (GE Healthcare) were used for the detection of GAPDH.
3
GST-Mdm35 Mutants Interaction Assay
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GST-Mdm35 mutants (10 μM) in 20 mM Tris-HCl pH 7.5 and 150 mM NaCl and 10 μM of His6–Ups1–Mdm35 in 20 mM the same buffer were mixed and incubated with 50 μl of GS4B resin at 37 °C for 15 min. The resin was washed three times with 500 μl of 20 mM Tris-HCl pH 7.5, 150 mM NaCl and proteins were eluted with 50 μl 10 mM glutathione in 50 mM Tris-HCl pH 8.0. The eluted proteins were subjected to SDS–PAGE and immunoblotting with the anti-His tag antibody (GE Healthcare).
4
Isolation and Binding Analysis of PG
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Isolation of PG from Anabaena and transmission electron microscopy were performed as described in reference 8 (link), using trypsin instead of α-chymotrypsin. The in vitro PG binding assay was adapted from reference 48 (link). In brief, 50 µg of purified protein was centrifuged for 15 min at 4°C and 300,000 × g. The pellet was collected for analysis. The supernatant was mixed with 50 µl isolated PG layer and incubated for 20 min at room temperature. The mixture was centrifuged for 15 min at 4°C and 300,000 × g, the supernatant was collected for analysis, and the pellet was washed with 10 mM sodium phosphate (pH 6.8) and 500 mM NaCl. After centrifugation, the pellet and supernatant fractions were supplemented with Laemmli buffer up to uniform volume. To control for protein precipitation, the same procedure was done without addition of the PG layer. All samples were analyzed by Western blotting with an anti-His tag antibody (GE Healthcare, Munich, Germany). Transmission electron microscopy was performed using a Philips CM12 microscope.
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