161-0309 Prestn Std, High Range
Protein Unstained MW
myosin 200 kDa
phosphorylase b 97 kDa
BSA 66 kDa
ovalbumin 44 kDa
High Range Markers are provided in SDS-PAGE loading buffer and may be loaded directly into an SDS-PAGE gel. Allow markers to come to room
temperature before use. Load 1-5 μl per lane in a mini-gel system. Sample preparation procedures are provided for monolayer cells, suspension cells
and tissue samples. Follow the procedure suited to your needs.
■Grow cells to subconfluency in a 100 mm x 20 mm petri dish, remove culture medium and rinse cell monolayer with room temperature 1x PBS
(10X liquid PBS: sc-24946). The following steps should be performed on ice or at 4° C using fresh, ice cold buffers.
■Add 0.6 ml of RIPA buffer (sc-24948) to the monolayer cells in the plate. Gently rock plate for 15 minutes at 4° C. Remove adherent cells with a
cell scraper. Transfer the resulting lysate to a microcentrifuge tube.
■Wash plate once with 0.3 ml of RIPA buffer and combine with first lysate. (Optional: Add 10 μl of 10 mg/ml PMSF (sc-3597) stock and/or pass through
a 21-gauge needle to shear the DNA.) Incubate 30-60 minutes on ice.
■Centrifuge cell lysate at 10,000xg for 10 minutes at 4° C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microcentrifuge
tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernantants.
■Collect approximately 2.0 x 107 cells by low-speed centrifugation (e.g. 200xg) at room temperature for 5 minutes. Carefully remove culture medium.
■Wash the pellet with PBS at room temperature, and again collect by low-speed centrifugation. Carefully remove supernatant.
■Add 1.0 ml of ice cold RIPA buffer (sc-24948) with freshly added protease inhibitors and/or phosphatase inhibitors. Gently resuspend cells in RIPA
buffer with a pipet and incubate on ice for 30 minutes.
■Further disrupt and homogenize cells by hydrodynamic shearing (21-gauge needle), dounce homogenization or sonication, taking care not to raise
the temperature of the lysate. (Optional: Add 10 μl of 10 mg/ml PMSF stock (sc-3597). Incubate 30 minutes on ice.
■Transfer to microcentrifuge tube(s) and centrifuge at 10,000xg for 10 minutes at 4° C. The supernatant fluid is the total cell lysate. Transfer
the supernatant to a new microfuge tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume
of RIPA, centrifuge and combine supernantants.
■Weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue should be sliced very thinly and thawed in RIPA buffer
(sc-24948) containing protease inhibitors and/or phosphatase inhibitors. Use 3 ml of ice cold RIPA buffer per gram of tissue.
■Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator, maintaining temperature at 4° C throughout all procedures.
(Optional: Add 30 μl of 10 mg/ml PMSF (sc-3597) stock per gram of tissue.) Incubate on ice for 30 minutes.
■Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4° C. Remove supernatant and centrifuge again. The supernatant fluid is
the total cell lysate. A longer centrifugation may be necessary to obtain a clear lysate.
Store the unopened vial of Low Range Markers at -20° C. After thawing, store unused markers at 4° C.
STAINED MOLECULAR WEIGHTS OF CURRENT LOT
For research use only, not for use in diagnostic procedures.
Copyright © 2002 GENTAUR Molecular Products