This experiment describes the process of Sephacryl S-300 HR column chromatography of HeLa cell nucleus extract. This experiment is from the guide to protein purification and characterization by Houzhu Zhu.
Operation method
Sephacryl S-300 HR column chromatography of HeLa cell nucleus extracts
Materials and Instruments
HeLa cell nucleus extract Sephacryl S-300 HR Liquid Nitrogen TM buffer + 0.1 mol LKCl Move Materials and equipment For more product details, please visit Aladdin Scientific website.
XK26 40 column
HeLa cell nuclear extract (prepared from 12L cell culture, see pp.9l~93) (5-ml sample)
XK26/40 column (PharmaciaBiotech, Inc.)
Sephacryl S-300 HR (PharmaciaBiotech, Inc,)
Liquid Nitrogen
Reagents
TM buffer + 0.1 mol/L KCl
(For the recipe, see "Preparation of reagents", pp.131~138)
Operating procedure
All operations were performed at 4℃.
1) Take 5-ml HeLa cell nuclear extract sample (from 12L cell culture) for gel filtration chromatography, and reserve 50ul of the extract for subsequent viability analysis.
2) For the XK26/40 column, the following column parameters were used:
Diameter = 2.6 cm
Column length = 34 cm
Volume = 180 ml
Flow rate = 100 ml/h
Buffer=TM buffer+0.1mol/LKCl
3) Add 5-ml sample of HeLa cell nuclear extract to S-300 column. Collect 5-ml fractions in separate sections.
Note: The critical step in gel filtration chromatography is the entry of the sample into the packed bed. The sample must enter the gel as a thin, uniform disk. The packing is most uniform and dense at the bottom of the column. For this reason, some researchers prefer to invert the column before use and then load the column from the top of the column when this end is the bottom of the column. Another approach is not to place the column, but from the bottom of the column spiked sample, so that the sample upward, which is also feasible. In both cases, the sample is added at the end of the column where the packing is densest and most uniform. It should also be noted that, in order to achieve high resolution of chromatography, the size of the component should be obtained smaller, generally about half of the upper sample volume. 
4) A 50-ul aliquot of each fraction eluted from the S-300 column is reserved for DNAzyme I footprinting analysis. The transcription factor AP-1 will elute at a Ve/V0 value of about 1.3~1.35. Many other transcription factors such as Spl, NF-I/CTF, and AP-2 will elute at a Ve/V0 value of approximately 1.35 to 1.40 (see Figure 2-2, p.95 for a typical elution diagram of an S-300 column).
5) The components can be stored at 4°C if used within 24 hours, or frozen in liquid nitrogen and stored at -80°C for long term storage.
Note: The freezing and thawing of protein solutions is one of the most important aspects of successful protein chemistry. There are several ways to do this. The authors recommend that protein samples should be frozen by immersion in liquid nitrogen as soon as possible (dry ice/ethanol will also suffice). However, the authors do not recommend placing protein samples directly into a -80°C freezer. In addition, it is of utmost importance that frozen protein samples be thawed as soon as possible. Loss of protein activity is often caused by thawing, which is often a very fast process as opposed to freezing. The authors recommend that frozen protein samples be thawed as quickly as possible by immersing them in room temperature water, and it is also important to remove any ice that may have formed around the container in order to speed up the thawing process.
6) Determine the DNA binding activity of AP-1 in each fraction by DNAzyme I footprinting (sample results, see Figure 2-3, p.99).
7) The S-300 column fraction containing the AP-1 activity peak can be directly loaded onto a sequence-specific DNA affinity chromatography column (see Experiment 3).