Matt Gethers/CRI, Thailand/Labwork/HmgR Purification Protocol Using Heparin

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HmgR Purification Protocol Using Heparin Column

  1. Weigh out 0.5 g freeze-dried powder (gives about 2 ml final volume of medium) and suspend it in distilled water. The medium swells immediately and should be washed for 15 minutes with distilled water in a beaker.
  2. Decant the water off of the heparin and pour into a clean column.
  3. Wash the column with 50 ml high salt (1 M NaCl or KCl), 50 ml low salt (50 mM), then 50 ml water.
  4. Equilibrate the column with 50 ml low salt buffer (look under the buffer section for contents). Leave some buffer above the heparin so subsequent buffers don't disturb the surface of the powder when they drip.
  5. Add your sample to the column and collect the flow-through; if your protein doesn't bind the column, it will be in this fraction.
  6. After the sample has flowed through the column, use 50 ml low salt buffer with 1 mM DTT to wash the putatively bound protein. Collect the wash through.
  7. After washing, fill the mixing apparatus with 50 ml low salt buffer and 50 ml high salt buffer in separate containers. Be sure that mixing is taking place and start to elute the protein from the column. If you use 50 ml of each buffer and collect 3 ml per fraction, you should use about 28-30 tubes.
  8. After elution, clean the column with 50 ml high salt, 50 ml low salt, and 50 ml water, then add 20% EtOH to preserve the column.


Buffers

Low Salt Buffer (500 ml)

Reagent Volume Final Concentration
5 M NaCl 5 ml 50 mM
500 mM EDTA 1 ml 1 mM
1 M DTT (Don't add to master) 1 mM
1 M Tris pH 7 25 ml 50 mM
100% Glycerol 25 ml 5%
H20 444 ml

High Salt Buffer (50 ml)

Reagent Volume Final Concentration
5 M NaCl 10 ml 1 M
500 mM EDTA 0.1 ml 1 mM
1 M DTT 50 μl 1 mM
1 M Tris pH 7 2.5 ml 50 mM
100% Glycerol 2.5 ml 5%
H20 ~35 ml (up to 50 ml)

Run Notes

7.11.08

Ran the protocol as written to bind and elute HmgR. Eluted in 28 fractions. Using a 1 in 2 dilution of 5x Bradford stock and 50 μl per sample, I used a 96 well plate to assay each fraction for protein. Fractions 3:8 and 13:19 had noticeable peaks after adding 150 μl of sample. I prepared the following samples for a poly-ac gel: BL21 lysate supernatant (lysed cells expressing HmgR from 200 ml culture), BL21 lysate pellet, column flow through, column wash through, and fractions 3, 5, 6, 7, 9, 11, 13, 14, 15, 16, 18, 20. The fractions were all made up with 15 μl fraction, 3 μl SDS loading buffer while the other samples used 100 μl sample and 20 μl loading buffer. 10 μl were loaded on a poly-ac gel and the results are recorded here. I pooled fractions 14, 15, 16, and 17 (~12 ml total) and dialyzed over night. On Saturday morning, I poured the sample into a tube, placed on ice, and placed in the 4oC until Monday.

7.14.08

Ran the protocol as written to further purify HmgR, but I used a Q-sepharose column to utilize ion exchange. Still eluted in 28 fractions using high and low NaCl buffer. Rather than preparing Q-sepharose, though, Mayuree allowed me to use a column she has in the 4oC that was stored in 20% EtOH. I started by washing the column in high salt, low salt, and water, then I followed the protocol as written. Using a 1 in 2 dilution of 5x Bradford stock and 50 μl per sample, I used a 96 well plate to assay each fraction for protein. Fractions 16:20 formed a noticeable peak after adding 100 μl of sample. I prepared the following samples for a poly-ac gel: Pooled fractions 14 through 17 (purified on 7.11.08, not yet run on the ion exchange column), q-sepharose flow through, q-sepharose wash through 1 (1st 10 ml), q-sepharose wash through 2 (remaining wash), then fractions 16, 17, 18, 19, and 20. All samples were made up with 15 μl sample and 3 μl SDS loading buffer. 15 μl were loaded on a poly-ac gel and the results are recorded here. I pooled all the fractions loaded on the gel (16:20) (~15 ml total) and dialyzed over night.

7.28.08

Resuspended each of 8 pellets (6 from 200 ml culture yesterday, 2 from samples of the culture at different times) in 1 ml resuspension buffer (10 ml Low salt buffer + 10 μl 1 M DTT + 1 Protease inhibitor cocktail tablet) and sonicated each. I spun down the lysate first for 5 minutes at 6,000 RPM, then for 5 minutes at 10,000 RPM (supernatant looked pretty turbid after first spin). I pooled the supernatants, but I'm keeping the pellets to run on a gel later today. Simultaneously, I washed the heparin column (made 7.11.08) with 1 M NaCl, 50 mM NaCl, and water. Then I equilibrated in low salt buffer without DTT. I added the supernatant sample to the column and collected the flow through, then washed with low salt buffer and collected the wash through in two fractions (1st 10 ml and the rest). I then did the elution with low and high salt; collected 28 3 ml fractions and placed on ice.

Bradford results here.

SDS-PAGE results here.

References

Heparin Sepharose Protocol