The control sample (FSEM containing DMP1) was added. Antibody staining was done using the primary antibody (LF 148 or Takkarra Inc., San Jose, CA, USA). The secondary antibody (Anti-rabbit IgG, Sigma-Aldrich, Burlington, Massachusetts, USA) was used. According to the manufacturer's manual, the antibody-positive fractions containing DMP1 were pooled and concentrated using Amicon Ultra-15 Filter Devices (Millipore 10,000NMWL). BCA protein Assay Kit (Pierce, Rockford, Illinois, USA) was used to quantify the amount of protein present in the pooled fractions by measuring the absorbance at or near 562 nm on a plate reader.
Amicon ultra 15 filter device
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra-15 Filter Devices are laboratory equipment used for the ultrafiltration and concentration of biological samples. The devices employ a membrane-based filtration system to separate and concentrate molecules of interest from complex mixtures, based on their molecular weight.
Automatically generated - may contain errors
6 protocols using amicon ultra 15 filter device
1
Immunodetection of Dentin Matrix Protein 1
2
Passive Immunization Against P. aeruginosa
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Mice were given IgG-enriched antiserum (100 μL IP, 400 μg IgG/mouse), or naïve serum, 4 hours prior to the challenge with PA14. IgG-enriched antiserum was prepared using purified antibodies obtained from IN- and IM-immunized mice using the previous vaccination regimens (Figs 1A and 4A ), which were then diluted in naïve serum. Antibody purification was carried out using the Protein G GraviTrap gravity-flow columns (GE Healthcare Life Science, Inc., USA). Amicon Ultra-15 filter devices (Millipore, Inc., USA) were used thereafter for desalting and concentration of eluted antibodies.
3
Optimizing K279a Protease Activity
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We have previously shown that Dulbecco's modified essential medium (DMEM) low glucose (5.6 mM) medium (Invitrogen) is the optimal growth medium for inducing K279a protease activity (20 (link)). To prepare a stock solution of K279a CS, 10 μL of an overnight K279a culture was inoculated in 6 x 15 mls of DMEM low glucose (5.6 mM) medium and grown for 48 h at 37°C on an orbital shaker. K279a CS was passed sequentially through 0.45-μm and 0.2-μm filters millex filters (Millipore Corporation, Bedford, MA). Culture supernatant (90 mls) was then concentrated using 5-kDa nominal-weight limit (NMWL) cut-off Amicon® Ultra-15 filter devices (Millipore Corporation, Bedford, MA). All concentrates were centrifuged at 4,000 × g and subsequently diafiltered by centrifugation with sterile DPBS to remove any low molecular weight contaminants including glucose and amino acids present in DMEM. An equivalent volume of DMEM was used as a negative control and for correction during protein quantification using the BCA (bicinchoninic acid) assay.
4
HIV-1 BaL Strain Growth Protocol
5
HIV-1 BaL Strain Propagation
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HIV‐1 BaL strain (cat #510 from NIH AIDS reagent program) was used in all experiments. They were grown in phytohaemagglutinin (PHA)‐stimulated CD8‐depleted peripheral blood mononuclear cells as described previously.33 The virus‐containing cell supernatant was filtered using an amicon ultra‐15 filter device (Millipore, Billerica, US) to remove the soluble cytokines. The residual levels of cytokines were tested using MSD and were found to be below 10 pg/mL. The control culture supernatant was prepared from uninfected cells in similar fashion.
6
Quantifying Draxin-AP Binding in Neurons
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Control-AP (pAPtag-5 vector) and draxin-AP constructs were transfected into HEK293T cells (Riken BRC)12 (link). After 4– 5 days, the conditioned media was harvested and then concentrated with the Amicon Ultra-15 filter device (Millipore). In the conditioned media, draxin-AP was detected using western blotting with an anti-draxin antibody (1:1,000)12 (link). Control- and draxin-AP concentrations in the conditioned medium were determined with the SensoLyte pNPP Secreted Alkaline Phosphatase Reporter Gene Assay Kit (AnaSpec). Draxin-AP binding to the dissociated neurons and brain sections was investigated as previously described12 (link)65 (link). We quantified draxin-AP signals on the growth cones of dissociated neurons from the neocortex, anterior dorsal thalamus and posterior dorsal thalamus. For the visualization of draxin-AP binding, neurons were stained with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium for 12 or 24 h at room temperature. We calculated the normalized AP signal intensity, which was the signal intensity at the growth cones of dissociated neurons divided by the signal intensity of the background. In each condition, AP signals were measured for three neurons using the BZ-II analysis system (Keyence) and mean values were calculated. Statistical analyses were performed on the data obtained from three independent experiments.
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