Preparation of crude TFIID, GAGA factor and RNA polymerase  fractions from the Elgin nuclear extract using the FPLC and DEAE and Phosphocellulose chromatography (8/14/91, updated 1/24/99).

Overview: Two Elgin nuclear extracts, each from approximately 100 grams of embryos are used (you can use less but scale the columns sizes appropriately). This will total about 200 mg of protein. The embryos are collected every 12 hours over a period of 3 days and stored on the grape plates in the cold room until preparation of the nuclear extract on the third day. The Elgin nuclear extract, which has been dialyzed to an HGKEDPX concentration between 100 and 200 mM, is flash frozen in liquid nitrogen and stored at -75oC prior to fractionation.  for my most recent preparation (Spring 97), I only fractionated the extract on DEAE cellulose and skipped the phosphocellulose column.  Good TFIID binding activity could be immunoprecipitated from the 0.25 M DEAE fraction.  GAGA factor is present in the DEAE flowthru.  RNA polymerase II is present in the 0.6 M DEAE fraction

The success of the fractionation requires that the columns be fully equilibrated immediately before loading the sample. Partial equilibration can be performed the day before but pH and conductivity change with storage of both ion exchange columns. The conductivity and pH of the column eluant should be tested immediately prior to loading the proteins to insure proper equilibration. DEAE column should not be reused because they tight bind nucleic acids which may alter the chromatographic properties on subsequent runs. Phosphocellulose can be reused after a 2M HGKE wash.

The two, thawed nuclear extracts are combined and adjusted to a 0.1M HGKEDP with OM HGKEDP (see end for description of HGKEDP buffers). This is loaded on a fresh, 60 ml DEAE column that has been previously equilibrated with 0.1M HGKEDP, washed with 0.1M HGKEDP. Proteins are then eluted with steps of 0.25M HGKEDP and then 0.6M HGKEDP. At this stage the flowthru and each salt step can be flash frozen and stored at -75oC. The flowthru contains most of the GAGA factor, which appears to be sensitive to slow freezing. The 0.25 M step contains the TATA complex. The 0.6 M step contains RNA polymerase II.

If the TFIID is to be further fractionated on phosphocellulose, the 0.25 M step containing the TFIID fraction is adjusted to 0.25M HGKEDP with 1M HGKEDP and then slowly loaded onto a 6 ml phosphocellulose column that has been previously equilibrated with 0.25M HGKEDP. The column is washed with 0.25M HGKEDP and the TFIID is eluted with 0.5M HGKEDP. Assess conductivity and protein concentration, aliquot into small portions, flash freeze, and store at -85oC.

Fractionation of the Elgin Nuclear extract on DEAE. (I recommend that you get the column equilibrated and the superloop filled the day before you want to run the column. Re-equilibrate just before applying sample.)

Preparation of DEAE according to the Whatman "preferred" method.

  1. Suspend 60 grams of resin in 360 ml of 0.2 M HEPES pH7.6 at room temperature.
  2. Stir with glass rod and adjust slurry to pH 7.6 with HCl. Settle and decant fines.
  3. Resuspend in 400 ml of 0.1 M HGKEDP. Stir with glass rod and adjust slurry to pH 7.6 with HCl. Settle and decant fines.
  4. Resuspend in 400 ml of 0.1 M HGKEDP. Stir with glass rod and adjust slurry to pH 7.6 with HCl.
  5. Pack a 60 ml column (settled by gravity at room temperature) into a 2.5 cm diameter amicon column. The excess resin can be stored for future use in the refrigerator.
  6. Cap the outlet and carefully attach the upper flow adaptor so that most of the air is expelled from above the resin. The flow adapter should be inserted until is contacts the bed of resin.
  7. Attach the column to the FPLC in the cold box. With the MV7 valve in the 3 position and the outlet of the column capped, attach the inlet of the column to port 1 and the outlet to the UV detector.
  8. Use the wash cycle to charge pumps A and B respectively with fresh, cold, 0M HGKEDP and 1M HGKEDP.
  9. Change MV7 valve to 1 and wash the column at 1ml/min. with 0.1M HGKEDP until it has equilibrated. The resin will pack down and the top flow adoptor will have to be inserted further to contact the bed. To do this, stop the flow and change the valve to position 3. Disconnect the outlet from the UV monitor and cap it with a nut. Next, disconnect the inlet from the valve. Remove the column from the cold box and screw the flowadaptor down against the packed bed. Reattach the inlet to position 1 and then attach the outlet to the UV monitor. Change the valve to position 1 and continue equilibrating at 1 ml/min. If a small space forms above the column of a few millimeters, the column is still adequate.
  10. During the column equilibration, attach and fill the 50ml superloop with 0.1M HGKEDP. Use the P1 pump to fill the superloop with buffer.
Fractionation. (The FPLC can be run in a fully automated mode or in a manual mode to achieve the described fractionations.)
  1. Charge the FPLC pumps with fresh buffer and the superloop with fresh 0.1M HGKEDP. Re-equilibrate the column if necessary. Set the UV monitor to the 2 absorbance scale.
  2. Thaw the nuclear extracts from a total of about 200 grams of embryos in an ice water slurry. This will be about 200 mg of protein.
  3. Combine extracts, determine conductivity and adjust the mixture to 0.1M HGKEDP with 0M HGKEDP.
  4. Centrifuge mixture for 15min. at 10,000 rpm in the HB4 rotor.
  5. Change MV7 to 1 and load cleared extract into the superloop with P1 pump.
  6. Set fraction collector to collect 24 min fractions.
  7. Change MV7 to 2 and inject the sample at 0.5 ml/min onto the column with 0.1M HGKEDP. Collect 12 ml fractions. After all of the sample has been injected onto the colum, switch the MV7 to 1 and wash the column with 100 ml of 0.1M HGKEDP at 0.5 ml/min; continue to collect fractions.
  8. Increase flowrate to 1 ml/min, set the fraction collector to collect 12 min fractions and begin eluting proteins with 0.25M HGKEDP. Plan on eluting with 150 ml of 0.25M HGKEDP. If the Abs280 of the eluate drops back to the base line with less than 150 ml, proceed to the 0.6M HGKEDP step.
  9. Increase salt to 0.6M HGKEDP and elute proteins with 150 ml of 0.25M HGKEDP.
  10. Identify the protein peaks by determining the protein concentrations of individual fractions with the Biorad assay. For a given peak, pool the fractions that are within 25% of the fraction with the highest protein concentration. Generally, 3 fractions are pooled for each of the salt steps. The 280 absorbance for the 0.6M peak will exceed the 0.25M peak, but the Biorad protein assay indicates that it has less protein. GAGA is in the flowthru (total protein, 40 mg), TATA is in the 0.25M step (total protein, 100 mg) and RNA polymerase II is in the 0.6M step (total protein, 20 mg).
  11. Set aside in a separate tube a small portion of each pool for future assays. Flash freeze fractions in liquid nitrogen and store at -85oC.
  12. The DEAE resin is discarded since nucleic acids remain tightly bound to the resin.
Fractionation on phosphocellulose. Note that this is probably not necessary for experiments involving immunoprecipitation of TFIID.

The phosphocellulose column has been a major problem because the pH and conductivity quickly changes when the column is not in use. As long as you a dilegent about making sure the pH and conductivity of the column is correct just before loading the sample on the column, new batches will work. Prepare the phosphocellulose material as described by the manufacturer. This is a full day's work, since the phosphocellulose is very dirty. For a DEAE fraction that originated from 100 g of embryos:

  1. A 6 ml phosphocellulose column is packed and it can be reused. It should be washed with 12 ml of 2M HGKE and then equilibrated to 0.25M HGKEDP before use. Flowrates should not exceed 0.5 ml/min. or the column may collapse.
  2. Charge the pumps with fresh buffer and the superloop with fresh 0.25M HGKEDP.
  3. Thaw the 0.25M DEAE fraction and adjust its conductivity with 1M HGKEDP to 0.25M HGKEDP.
  4. With MV7 valve in the 1 position, load protein into the superloop.
  5. Set absorbance scale at 2.
  6. Change MV7 to 2 and inject sample at 0.15 ml/min onto the column with 0.25M HGKEDP. Collect 20 min. fractions (3ml). After all of the sample has been injected onto the colum, switch the MV7 to 1 and wash the column with 18 ml of 0.25M HGKEDP at 0.15 ml/min.
  7. Increase salt to 0.5M HGKEDP and elute with 18 ml at 0.15 ml/min. Collect 3 ml fractions. This should contain the TATA complex.
  8. Increase salt to 1M HGKEDP and elute with 18 ml at 0.15 ml/min.
  9. Identify the protein peaks by determining the protein concentrations of individual fractions with the Biorad assay. For a given peak, pool the fractions that are within 25% of the fraction with the highest protein concentration. Generally, 3 fractions are pooled for each of the salt steps. Determine conductivity and protein concentration. Flow thru contains 50mg; 0.5M step contains 16mg; and the 1M step contains 6mg.
  10. Aliquot 0.5M step into numerous small portions. One to three microliters should be adequate for footprinting, crosslinking, or gel shifting. Although the TFIID will tolerate one or two freeze/thaw cycles, there is a drop in activity after numerous cycles. Flash freeze fractions in liquid nitrogen and store at -75oC.
Column Buffers:
HGKEDP buffer contains 25 mM HEPES pH 7.6 (adjusted with NaOH), 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and potassium chloride as indicated (ie. 0.5M HGKEDP contains 0.5M KCl). The DTT, and PMSF are added just before use from 1M and 100mM stocks, respectively. Prepare 1 liter of 2X OM HGKE (50 mM HEPES pH 7.6, 20% glycerol, 0.2 mM EDTA) and 1 liter of 2M HGKE (2 M KCl, 25 mM HEPES pH 7.6, 10% glycerol, 0.1 mM EDTA). Filter each stock through nitrocellulose, the 2M HGKE is particularly dirty.

1 liter 2X OM HGKE (note that this is 2-fold concentrate):


1 liter 2M HGKE

Prepare needed amounts of 0M HGKEDP and 1M HGKEDP from these stocks. Don't forget to dilute the stocks or the columns will not run as expected.