Krause lab protocols
Affinity chromatography (with HIS-tagged proteins)
This protocol is described in less detail in Copeland et al, Nature, 379, 162-165, 1996.
Affinity chromatography can be performed using a number of different protein tags. In our lab we have had success using poly-hisitidine tagged proteins for creation of our protein affinity columns. The histidine tag is very short (10 his residues) and should not alter the conformation of the tagged protein, nor should it be involved in artifactual interactions. The poly-his tag binds to a nickel chelate resin for creation of the column. We use the resin alone for the control columns. Most often, we challenge our columns with 35S-labeled, in vitro translated proteins, however, proteins from a variety of sources can be used and detected appropriately.
The protocol below describes our method. The order of the steps has been optimised so that both the affinity columns and in vitro translated protein are ready at the same time.
1) Begin by getting out the DNA and 35S-methionine for the in vitro translation reactions.
2) While these thaw, prepare solutions for affinity columns: AC Buffer: (10% glycerol, 100mM NaCl, 20mM Tris pH 7.6, 0.5mM EDTA, 0.1% Tween-20, ddH2O).
NOTE: You may wish to add protease inhibitors (eg. TPCK), but usually we don't; you may also choose to filter sterilize the buffer, but again, usually we don't.
5% milk powder solution: 10 mL AC Buffer + 0.5g milk powder. Mix on a shaker platformuntil dissolved.
Store both solutions on ice.
4) Prepare the nickel resin. 50uL (settled volume) of resin is required for each experimental and control column. Transfer the appropriate volume of slurry into two siliconized eppendorf tubes, one for the control resin, one for the protein coupling. The resin should be allowed to settle and then washed once in water and twice in AC buffer. In each wash step the resin can be allowed to settle by gravity or by centrifuging at 1.5K for 3 minutes in a benchtop centrifuge.
5) Between washes of the resin begin to set up the in vitro translation reactions for the proteins that are to be tested for binding. We follow the standard protocol for a 25uL reaction using the Promega TNT coupled transcription/translation kit.
6)While the reactions are incubating couple your protein to the resin. We use 1mg of pure His-tagged protein per mL of resin. However, the optimal ratio may vary depending on the interaction being pursued. We treat the control resin with the same buffer that the protein is stored in. Allow the coupling to proceed for 30 minutes on ice.
7) Prepare spin columns for removal of unicorporated 35S-methionine from the translation reaction. The columns are made in 1 mL syringes blocked with glass wool. The resin is G-25 (BioRad) equilibrated in AC buffer. Follow the Maniatis protocol for spin columns.
NOTE: in the following steps, you want to avoid foaming and bubbles as much as possible, as the bubbles can contribute to protein denaturation. So, always mix gently.
8)While the protein is being coupled, and between spins in the preparation of spin columns, begin to set up the housings for the affinity columns. We use 200uL tips with bevelled ends (Diamed #230-1673-03K). The tips have to be blocked with glass beads to prevent the resin from leaking out. Use Sigma Acid washed 212-300 micron glass beads that have been washed in water. Beads should be added to a level of 7mm at the end of the tip. After the beads have been transferred, remove excess water and then add 100uL of AC buffer. This is to prevent the contact of protein resin directly with water.
At this time the fraction collection tubes can also be labeled. The columns are run standing in eppendorf tubes on ice, the columns are moved to new tubes for each fraction. 7 collection tubes will be needed for each column: 1 flow-through fraction, 4 wash fractions, and 2 elution fractions.
9) When the in vitro translation reactions are finished pass the reaction over the spin column in a volume of 100uL.
10) Set up the column load sample. 50uL in vitro reaction (half the sample after the spin column), 130 uL AC buffer, and 45uL 5% milk powder. Total volume=225 uL
11)Wash the nickel resin. After the protein has been allowed to couple to the resin for 30 minutes on ice, wash the resin twice in 1mL of AC buffer to remove unbound protein and to equilibrate the resin in AC buffer.
12) Set up the columns. Add 50uL of settled affinity resin to the prepared tips. Allow the resin to settle on ice.
13) Load the columns. After the resin has settled (5-10 minutes) remove any buffer remaining on top. Carefully load 90uL of the load sample to the top of each column (control and experimental). DO NOT DISTURB THE SETTLED RESIN. The remaining portion of the load sample should be saved for SDS-PAGE.
14) Allow the loaded protein to flow into the resin. When it reaches the bottom of the column (you can follow its progress because of the red color of the retic. lysate) transfer the column into tube #1. Allow the remaining protein to flow into the tube. This comprises the majority of the flow-through fraction. When the last of the load has flowed into the column transfer the column to tube #2.
15) Wash the columns with 4x400uL of AC buffer. Each wash fraction is 400uL. Transfer the column to a new tube after each wash step.
16) Elute the bound protein. After the last wash transfer the columns to tube #6 at room temperature. Add 30uL of 2% SDS (this would precipitate on ice). Allow this to flow-in. Transfer the column to tube #7. Add 120uL of 2% SDS. Allow this to run through. This is the eluate fraction.
17) For gels: Load and Flow Through will have 9 uL sample + 3 ul 4X SDS gel loading buffer
To compensate for dilution, eluate loaded will equal 15 ul sample + 5 uL 4X SDS
18) Boil the samples, store in freezer until ready for SDS-PAGE. After electrophoresis, dry the gel and autoradiograph. An interaction is detected as depletion of the labeled protein from the flow-through and presence of the protein in the experimental column eluate.
Affinity chromatography with FTZ (1st 3 lanes) and control (2nd 3 lanes) affinity columns. 35S-labeled PRD and FTZ-F1 bind to the FTZ columns and can then be eluted (E). (L=load, FT=flowthrough; Luc=luciferase, negative control).
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