Editing Phosphocellulose Chromatography

<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">Whatman P11 Cation Exchange Resin</span>

<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">or&nbsp;</span>Cellulose phosphate<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">&nbsp;</span>https://www.sigmaaldrich.com/catalog/product/sigma/c2258<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;"></span>

or&nbsp;

== <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">Generation</span> ==

<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;"></span>1. Stir the weighed cellulose (1 g will give approximately 5-6 ml swollen phosphocellulose) into 25 volumes of 0.5 N NaOH and leave for 5 min.

2. Decant the supernatant, add water and wash the cellulose in a funnel until the filtrate pH is 11 or below.

3. Transfer cellulose in fresh beaker and suspend in 25 volumes of 0.5 N HCl and leave for 5 min.

4. Filter the supernatant and wash in a funnel until the filtrate pH is above 3.0.

5. Suspend cellulose in 25 volumes of 10X equilibration buffer and incubate for 5 min with gentle agitation. Filter or decant supernatant away and repeat this step once again.

6. Suspend the cellulose in 25 volumes equilibration buffer, agitate for 5 min and filter. After all the buffer has almost passed through the cellulose, add fresh buffer and continue the filtration until another 25 volumes have passed through.

7. Check the pH of the filtrate. It should be very close to that of the equilibration buffer. If not, continue adding fresh buffer to the filter.

8. Suspend cellulose in equilibration buffer and pour it in a column (the column should be 20% filled with equilibration buffer). After 15 min start the flow of the column (not too fast) until all the material is packed. Increase the flow rate to approximately 150% of the flow rate to be used during the protein separation stages and run the column for an hour before sample application. Make sure that the pH of the eluate is that of the equilibration buffer before adding your sample (otherwise you might be heading towards a disaster).



== Magnetic DNA affinity beads  ==

Wash 0.005; 0.01; 0.015; 0.02 mg) of streptavidin magnetic beads (Streptavidin MagneSphere Paramagnetic Particles, Promega)&nbsp; twice with 0.5 SSC. 

Add Biotinylated oligonucleotide (100 ng)&nbsp; to the beads in 1TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0) and place&nbsp; in a roller at room temperature for 30 min


[[Category:Protein Purification]][[Category:Protocols]]