Approximately 10 × 106 LS174T colon cancer cells were collected by gentle cell scraping. Cells were diluted with 200 μl 1X PBS supplemented with 5 mM calcium and magnesium chloride and evenly split into two fractions. One fraction received buffer only and one received 1.5 U/ml GPI-specific phospholipase C (Invitrogen) for 1 hour at 37°C. The cells were collected by centrifugation and the supernatants were collected for analysis. Biotin labeled alpha toxin 2 μg/ml, was added and the samples were incubated at room temperature for 30 minutes. Streptavidin magnetic beads (Promega) were added for 30 additional minutes at room temperature. Beads were captured on a magnetic stand and washed 3X with 1X PBS before releasing the proteins with Laemmli buffer. Proteins were separated on a 4–12% Bis-Tris polyacrylamide gel (Invitrogen) and transferred to PVDF for detection of CEA5 (monoclonal antibody COL-1, Invitrogen) or gels were fixed and stained with silver.
Gpi specific phospholipase c
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
GPI-specific phospholipase C is an enzyme that catalyzes the hydrolysis of glycosylphosphatidylinositol (GPI) anchors from proteins. It is used in laboratory settings to release GPI-anchored proteins from cell membranes for further analysis and experimentation.
Automatically generated - may contain errors
2 protocols using gpi specific phospholipase c
1
Isolation and Detection of CEA5 from Colon Cancer Cells
2
Synovial Tissue Preparation for Rheumatoid Arthritis
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Synovial tissue samples were obtained by needle arthroscopy from patients with RA [15 (link)] who fulfilled the American College of Rheumatology/European League Against Rheumatism classification criteria [16 ]. The Medical Ethics Committees of the Academic Medical Center and Medical University of Vienna approved the study, and all patients gave written informed consent. Biopsy samples were snap-frozen in Tissue-Tek OCT (Miles Inc, Elkhart, IN, USA) immediately after collection. Cryostat sections (5 μm) were cut and stored at −80°C. Alternatively, synovial tissue obtained from synovectomy or joint replacement surgery was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated, paraffin-embedded, sectioned at 4-μm thickness, and stored at room temperature.
To remove GPI-linked proteins, thawed sections were washed two times in PBS and treated with 1 U/ml GPI-specific phospholipase C (Invitrogen, Bleiswijk, Netherlands) for 45 min at 37°C. Then, sections were fixed in acetone, washed with PBS, and stained as described below.
To remove GPI-linked proteins, thawed sections were washed two times in PBS and treated with 1 U/ml GPI-specific phospholipase C (Invitrogen, Bleiswijk, Netherlands) for 45 min at 37°C. Then, sections were fixed in acetone, washed with PBS, and stained as described below.
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