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I want to purify crude alpha-amylase with column chromatography. I am using a spehadex 75, But for some reason I can't find any information on how to make the slurry.

I can quickly find tons of information on how to pack and store a column, but what i really want to know is how much powder and buffer do I mix together?

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The '75' of Sephadex G-75 refers to the water regain value, which is defined as the grams of water absorbed upon hydration of 1g dry powder, multiplied by 10 (see Reiland). Thus the water-regain value of Sephadex G-75 is about 7.5ml. This value refers only to the water contained within the gel particles and not to the water trapped outside of the particles in a packed gel (Reiland). As a rule-of-thumb, the latter figure can be estimated to 50%.

So ... 1g of dry Sephadex will give about 15 ml of packed gel

From here: 1 g swells to 12-15 mL gel

Just a thought ... Does alpha-amylase interact with Sephadex? Maybe 'thinking' it resembles a substrate, and (reversibly) binding to it or otherwise interacting to cause effects in addition to gel-filtration ?

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