Operational experiments in affinity chromatography
Operational experiments in affinity chromatography
This experiment introduces the related operation of affinity chromatography. This experiment is from the Laboratory Guide for Protein Purification and Identification, by Zhu Houzhu.
Operation method
Operational experiments in affinity chromatography
Materials and Instruments
Non-specific competition DNA Liquid Nitrogen Buffer Z Buffer Ze Column Regeneration Buffer Column Preservation Buffer Move Materials and equipment For more product details, please visit Aladdin Scientific website.
EconoCoIumn
EconoCoIumn column. (Bio Rad Laboratories731-1550)
Non-specific competition DNA
Liquid Nitrogen
Reagents
Buffer Z
Buffer Ze
Column regeneration buffer
Column Preservation Buffer
(For formulations, see "Reagent Preparation," PP.131-138)
Operating Procedures
The following experiments were performed with Buffer Z, but good results can also be obtained with TM buffer. It is important to note that some commercially available HEPES and PIPES buffers contain contaminants that inhibit the binding of proteins to DNA, and therefore TM buffer is safer in some respects. Some transcription factors, such as SP1, have a higher affinity for DNA in the absence of Mg2+ than in the presence of Mg2+. Therefore, it may be advantageous not to use Mg2+ in chromatography buffers, e.g., Ze buffer is identical to Buffer Z except that it does not contain MgCl2. Although, in the procedure described herein, the chromatography buffer contains KCl, KCl can mostly be replaced with NaCl. For purification of AP-1, either Buffer Z or TM Buffer can be used.
Note: DNA affinity chromatography is generally performed at 4°C. However, it has been observed that some sequences have been purified. However, it has been observed that some sequence-specific DNA binding factors bind DNA (and DNA affinity media) with an affinity higher than 4°C at room temperature approximately (22°C.) Therefore, for each factor, it is important to determine its optimal binding temperature.
1) Equilibrate DNA Affinity Medium (1 ml bed volume) loaded on a 2-ml Bio-Rad Econo column with 2X10 ml of Buffer Z+0.1 mol/L KCl.
2) Protein fractions (dissolved in TM Buffer + 0.1 ml/L KCl, Buffer Z + 0.1mol/L KCl, or Buffer Ze + 0.1mol/L KCI) are mixed with non-specific competitive DNA.
Note: For purification of AP-1, 0.51 ml of sonicated poly(dI-dC) at a concentration of 10A260 nm was mixed with approximately 25 ml of S-300 column peak eluate (in TM Buffer+1mol/LKCl) prepared from 12 liters of HeLa cells at a concentration of 10A260 nm poly(dI-dC) ≈ 850ug DNA /ml.
3) Protein-DNA mixture was allowed to stand for 10 min.
4) Centrifuge the mixture for 10 min at 10,000 r/min with an SS-34 turntable to precipitate the insoluble protein-DNA complex. Transfer the supernatant to a new tube.
5) Load onto the chromatography column by gravity flow (15 ml/h/column).
NOTE: One 1-ml column should be sufficient to purify AP-1 from 12L cells. Multiple 1-ml columns may be used in parallel to purify large quantities of material, however, typically up to 50 ml of protein sample can be loaded on a single 1-ml column.
6) After loading the raw material, wash the column with 4x2 ml of Buffer Z (or Ze) + 0.1 mol/L KCl.
Note: In this step, it is very important that the column be thoroughly cleaned; DNA affinity columns typically purify the factor to be isolated by more than 100-fold. However, when proteins are purified 100-fold, even 1% of heteroproteins in the raw material can become major impurities in the affinity chromatography purified transcription factors due to incomplete cleaning of the affinity column.
7) Proteins were eluted from the column with 1-ml buffer Z containing 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 mol/L KCl, and 3Xlml buffer Z containing 1 mol/L KCl, added sequentially. The 1-ml fractions corresponding to the 1-ml buffer portion added were collected in sections.
Note: When purifying a new factor, a step of elution with a salt solution containing 2 mol/L NaCl should also be included. This is because in a few instances it has been found that sequence-specific binding proteins can still bind to the affinity medium at a salt concentration of 1 mol/L (in these instances, the factors are eluted with 2 mol/L salt solution). When eluting with salt concentrations greater than 1 mol/L, NaCl is preferred over KCl because NaCl is more soluble than KCl.
8) Keep two aliquots of each component (40ul for SDS-PAGE analysis and 25ul for DNA enzyme I footprint analysis) at 4°C, and store the rest of the components at -80°C after freezing in liquid nitrogen.
9) Regenerate the affinity medium as follows: Stop the flow of each column at room temperature and add 5 ml of column regeneration buffer to each column. Stir the media with a fine siliconized glass rod to mix the media with the column regeneration buffer. Allow the buffer to drain out from under the column. After stopping the flow again, repeat the above steps of adding buffer and mixing it with the media. Then equilibrate with 2X15 ml of Column Storage Buffer and store the media at 4°C.
10) Analyze the protein fractions for sequence-specific DNA binding activity (see Figure 2-4 for 8% SDS-polyacrylamide gel electrophoresis silver staining of AP-1 purified by affinity chromatography). For further purification, the active fractions can be combined. The combined fractions are then diluted (with KC1-free buffer Z) or dialyzed (against buffer Z+0.1 mol/L KCl) to a final KCl concentration of 0.1 mol/L. The protein fractions are mixed with non-hostile competing DNA (the type and amount required is determined experimentally) and then purified by affinity chromatography on a column. 