161-0309 Prestn Std, High Range

PROTEIN MARKERS

Protein Unstained     MW

myosin                     200 kDa

phosphorylase b     97 kDa

BSA                           66 kDa

ovalbumin               44 kDa

PROCEDURE

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.

MONOLAYER CELLS

■ 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.

SUSPENSION CELLS

■ 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.

TISSUE SAMPLES

■ 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.

STORAGE

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

RESEARCH USE

For research use only, not for use in diagnostic procedures.