Procedure

1. Begin by cleaning the FPLC equipment:

a. Wash the gel-filtration column with at least 150 ml of gel-filtration buffer.

b. Wash the injection loop and the injection needle with gel filtration buffer.

All FPLC gel-filtration steps should be performed in a cold room or chromatography refrigerator at 4°C.

2. Prepare 1 ml of cold sample grinding buffer (gel-filtration buffer with freshly added 1 mM PMSF, 1 mM (3-mercaptoethanol). Grind plant material on ice using a pestle and mortar until a homogeneous sample is obtained.

3. Transfer the extract to a microcentrifuge tube and remove cell debris by centrifuging at 13,000 rpm for 5 minutes at 4°C in a benchtop centrifuge.

4. Filter the supernatant through a syringe filter (0.2-pM pore size).

5. Measure the protein concentration of the sample using standard methods such as the Bradford (1976) assay. The protein concentration should be in the range of 150-2000 pg/ml.

6. Inject 200 pi of the filtered supernatant (30-200 pg of total soluble protein) into the gel-filtration column.

7. Run a gel-filtration program that will discard the initial 7.5 ml of the eluate (exclusion volume of the Superose 6 and Superdex 200 columns), and then collect 30 0.5-ml fractions in microcentrifuge tubes. If necessary, separation can be improved by collecting more fractions of smaller volume.

Following gel filtration, the gel-filtration column must be washed with excess gel-filtration buffer before it can be used again.

8. For separation of the proteins on an SDS-PAGE gel and western blot analysis, concentrate the protein fractions by using either Strata-Clean beads or Microsep cartridges.

• Using StrataClean beads: Add 10 pi of StrataClean beads suspension to each 0.5 ml of protein fraction and incubate on a rotating sample holder for 10 minutes at 4°C. Collect the Strata-Clean beads by centrifugation at 13,000 rpm for 1 minute at 4°C in a benchtop centrifuge. Discard the supernatant and resuspend the pellet in 40 pi of protein gel-loading buffer. Denature the proteins by boiling the samples for 5 minutes and then quick-chill the samples on ice, followed by a brief spin to remove the Strata-Clean beads.

• Using Microsep cartridges: Concentrate the protein samples using Microsep cartridges to obtain a sufficiently small sample volume that allows SDS-PAGE analysis, e.g., 20 pi.

9. Carry out SDS-PAGE on the protein samples, followed by western blot analysis.

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