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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

GST Pull-Downs from Drosophila Tissue Culture Cells

GST Pull-Downs from Drosophila Tissue Culture Cells
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Procedure
A. Preparation of Glutathione-Agarose Beads (see Hint #2)

1. Prepare cellular extracts that contain the Glutathione-S-Transfers (GST) fusion protein (see protocol on Preparation of Cellular Extracts) in NETN with 1 M NaCl Buffer.

2. Following the manufacture's instructions, prepare the Glutathione-Agarose beads and when completed with the manufacture's instructions, wash the beads extensively in NETN with 1 M NaCl Buffer.

B. Protein Binding to Glutathione-Agarose Beads

1. Incubate an appropriate amount of Cellular Extract with the Glutathione-Agarose beads for 45 min to 1 hour with gentle rotation at 4°C.

2. Pour the Beads containing Cellular Extract slurry into a column of appropriate size for the bed volume of beads.

3. Allow the beads to settle in the column (see Hint #3).

4. Open the flow at the bottom of the column and allow eluate to flow to waste.

5. Wash the column with 30 column volumes of NETN with 1 M NaCl Buffer.

6. Wash the column with 40 column volumes of NETN with 100 mM NaCl Buffer.

7. Wash the column with 40 column volumes of the buffer in which the pull-down experiment will be performed (e.g. Reaction Buffer, see hint Hint #4)

8. After washing extensively, place the column of Glutathione-Agarose beads in the Reaction Buffer containing 100 μg/ml Bovine Serum Albumin on ice (or at 4°C, see Hint #5).

C. Pull-Down Experiment (see Hint #4)

1. Assemble the following components in a microcentrifuge tube on ice:

150 μl of 2X Reaction Buffer.

50 μl of Kc Nuclear Extract

20 μl of a 1:1 slurry of prepared fusion protein/Glutathione-agarose beads (prepared in Section B)

80 μl ddH2O

(see Hint #6)

2. Incubate the reaction at room temperature for one hour with rotation of the microcentrifuge tubes.

3. After incubation, all tubes and solutions should be kept on ice.

4. Centrifuge to pellet the Glutathione-Agarose beads in a microcentrifuge for 8 sec, and save the supernatant to a new microcentrifuge tube.

5. Wash away non-specific proteins by adding 500 μl of ice-cold Wash Buffer to the pelleted beads, mixing a few seconds by inverting tube a couple of times, and centrifuge to pellet the Glutathione-Agarose beads as in the previous step. Discard the supernatant.

6. Repeat Step #5 two more times.

7. Wash Glutathione-Agarose beads with 1 ml of ice-cold Wash Buffer as in Step #4 and discard the supernatant.

8. To assay the proteins, resuspend the Glutathione-Agarose beads in 2X SDS-PAGE Sample Buffer and boil samples 5 min.

9. Electrophoresis the boiled sample (from Step #6), the loaded supernatant (from Step #4) and starting extract (see protocol on SDS-PAGE of Protein Samples).

10. To assay nucleic acids, add 200 μl of 2X Proteinase K Buffer and incubate for 15 min at 30°C.

11. To the extract, add an equal volume of Phenol:Chloroform:Isoamyl Alcohol, mix well by vortexing, centrifuge in a microcentrifuge at maximum speed for 15 min to separate the phases and save the aqueous (upper) phase.

12. Repeat the Phenol:Chloroform:Isoamyl Alcohol (Step #11) extraction one more time.

13. To the aqueous phase, add an equal volume of Chloroform:Isoamyl Alcohol (49:1), mix well by vortexing, centrifuge in a microcentrifuge at maximum speed for 15 min to separate the phases and save the aqueous (upper) phase.

14. To the aqueous phase, add 0.1 volume of 3 M Sodium Acetate, 10 μg of Glycogen and 2.5 volumes of 100% Ethanol.

15. Mix well by vortexing and incubate overnight at -20°C.

16. Centrifuge in a microcentrifuge at maximum speed for 20 min to pellet the nucleic acids and discard the supernatant.

17. To the pellet add 500 μl of 70% Ethanol, mix well by vortexing, centrifuge in a microcentrifuge at maximum speed for 10 min to pellet the nucleic acids, and discard the supernatant.

18. Resuspend the nucleic acids in appropriate buffer and analyze by gel electrophoresis (see protocol on Analysis of DNA by Gel Electrophoresis or Analysis of RNA by Gel Electrophoresis).

Solutions
Chloroform:Isoamyl Alcohol   1% (v/v) Isoamyl Alcohol
49% (v/v) TE-saturated Chloroform (CAUTION! see Hint #1)
Store at 4°C
Phenol:Chloroform:Isoamyl Alcohol   Store in a dark bottle at 4°C under a layer of 0.1 M Tris-HCl, pH 8.0
1% (v/v) Isoamyl Alcohol
49% (v/v) Chloroform (CAUTION! see Hint #1)
50% (v/v) Phenol (CAUTION! see Hint #1)
TE Buffer   10 mM Tris-HCl, pH 8.0
1 mM EDTA.
Protein Sample Buffer (2X)   2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT
125 mM Tris
20% (v/v) Glycerol
0.001% (w/v) Bromophenol Blue
4% (w/v) SDS
0.1 M PMSF   in Isopropyl Alcohol
Store at -20°C
0.1 M Phenylmethylsulfonyl Fluoride (PMSF) (CAUTION! see Hint #1
Reaction Buffer (2X)   40 Units of RNasin RNase Inhibitor (40 Units total)*
40 mM HEPES-KOH, pH 7.6
600 mM KCl
6 mM ATP*
*Add just before use
20% (v/v) Glycerol
8 mM MgCl2
0.1% (v/v) Nonidet P-40 (NP-40)
6 mM Creatine phosphate*
NETN with 1 M NaCl Buffer   20 mM Tris-HCl, pH 7.5
0.5 mM PMSF*
0.05% (v/v) Nonidet P-40 (NP-40)
1 mM EDTA
*Add just before use
0.5 mM DTT*
1 M NaCl
2X Proteinase K Buffer   1% (w/v) SDS
2 mg/ml Proteinase K
Store in small aliquots at -20°C
1 M DTT   Store in small aliquots at -20°C
Wash Buffer   0.5 mM DTT*
0.1 mM EDTA
*Add just before use
4 mM MgCl2
0.05% (v/v) Nonidet P-40 (NP-40)
20 mM HEPES-KOH, pH 7.6
10% (v/v) Glycerol
300 mM KCl
0.5 mM PMSF*
NETN with 100 mM NaCl Buffer   0.5 mM PMSF* (CAUTION! see Hint #1)
0.05% (v/v) Nonidet P-40 (NP-40)
1 mM EDTA
0.5 mM DTT*
0.1 M NaCl
20 mM Tris-Cl, pH 7.5
 
BioReagents and Chemicals
2-Mercaptoethanol
Magnesium Chloride
Isoamyl Alcohol
Chloroform
Bovine Serum Albumin
Phenol
Proteinase K
PMSF
Glycerol
DTT
Creatine Phosphate
Potassium Chloride
Sodium Chloride
ATP
Tris Base
Nonidet P-40
Bromophenol Blue
EDTA
SDS
HEPES
RNasin
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Select the GST fusion protein of interest from bacterial extracts using Glutathione-Agarose beads. The amount of cellular extract that contains the GST fusion protein for a given bed volume of beads will depend on the expression level of the fusion protein and therefore must be determined empirically.

3. Make sure to have a column that is at least 10X the size in volume of the bed volume of Glutathione-Agarose beads. This will allow for an acceptable flow rate and for ease in adding large volumes of buffer. See Protocols on Column Preparation and Chromatography for specific instructions and considerations.

4. This section was specifically designed for Drosophila Kc tissue culture cell using nuclear extracts Kc NE.

5. Column can be stored this way for more than two weeks with daily changes of the final buffer

6. The components for this reaction have been empirically determined to comprise optimal reaction conditions for isolating small nuclear ribonucleoprotein complexes from Drosophila tissue culture nuclear extract. The nuclear extract is usually at a concentration of 5 to 10 mg/ml and there is approximately 1 to 2 μg of recombinant fusion protein on the beads. Experiment-specific conditions will need to be empirically determined.