PROTEIN BIOCHEMISTRY DAY TWO

Today, you will "desalt" your ammonium sulfate precipitated sample using a PD-10 gel filtration column, then test each fraction for B-gal activity using a "qualitative" B-gal assay. Sample(s) with B-gal activity will be pooled, and one half of the sample will be saved for PBC Day 3. The other half will undergo further fractionation on a DEAE anion exchange column.

During recitation, the staff will centrifuge your AS precipitation tubes, and will return them to you when you come to lab.	If your AS precipitation was successful, you should see a pellet (AS-P) on the side of the centrifuge tube.	In preparation for the desalting column, one partner should resuspend the ammonium sulfate pellet sample (AS-P).

The PD-10 column is shown here (foreground).	After loading your AS-P sample onto the PD-10 column, you will collect the flowthrough. Then, you will elute the column with column buffer. Setting up an Eppendorf rack just below your column makes it easy to collect fractions.

After collecting fractions of the PD-10 column, you will use a "qualitative" (by eye) B-gal assay to detect which fraction(s) have activity. These can be set up in either glass tubes (left photo) or Eppendorfs (center photo). If you have a hard time seeing the "yellow" color, place your tubes against a white background like your notebook (right photo).

While you are collecting and assaying fractions from the PD-10 column, the staff will come around with DEAE slurry to load into your empty column (left photo). Before equilibrating the column, you'll need to allow the slurry in the column to "settle." The center photo shows "unsettled" slurry, whereas the right photo shows "settled" slurry. Let the slurry settle undisturbed for 5-10 minutes before equilibrating the DEAE column.

After equilibrating your DEAE column, you will load your column and collect the flowthrough. After washing, you will elute the column with buffers of increasing NaCl concentration.

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