What is the function served by each ingredient in the fractionation buffer? You

Question

What is the function served by each ingredient in the fractionation buffer? You have just accomplished the feat of isolating nuclei from the ova of a "golden goose" The ideal condition for pelleting the nuclei involves centrifuging at 770 g for 10 minutes in a rotor of radius 11.02 cm. a. At what speed (in rpm) was the sample centrifuged? b. Your friend in California wants to repeat your procedure, but he has a rotor of radius 17.2 cm. At what speed (in rpm) should he centrifuge to pellet the "golden nuclei"?

Explanation / Answer

(1)The function of the ingradients used in fractionation buffer are as fllows -

(i) Salt - Salts like NaCl, KCl etc. are used to establish an ionic strength in the buffer solution.

(ii) EDTA- free complete tablet protease inhibitors are used to inhibit protease enzyme. This prevents the catabolism of protein by the hydrolysis of peptide bnd.

(iii) Dithiothreitol (DTT) is used as a reducing agent.

(iv) Distilled water is used to adjust pH.

(v) Buffers like HEPES and Tris etc. are sued as buffer.

(2) Here, radius, r = 11.02 cm = 0.1102 m

a = 770 g = 770 x 9.8 m/s2 = 7546 m/s2

Now, the formula for centripetal accleration of a centrifuge is

a = V2 / r

=> V = square root ( a x r)

=> V = square root(7546 m/s2 x 0.1102 m) = 28.84 m/s

(b) Again, r = 17.2 cm = 0.172 m

Now, in both cases the accleration should be same.

So,

a = 770 g = 7546 m/s2 = V2 / r

=> 7546 m/s2 = V2 / 0.172 m

=> V = square root (7546 m/s2 x 0.172 m) = 36.03 m/s

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