Gel filtration separates proteins.
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Last Update: 2020-10-30
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Source: Internet
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Author: User
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related topics .Objectives:
1, learning the principles and operating methods of gel
column
technology;
2, learning hemoglobin preparation methods.II, experimental principle:
1. Gel layering: principle and application
2. Principle of reaction to this experiment: Hemoglobin (hemoglobin, Hb) is the main inclusion of red blood cells, it is hemoglobin and globin peptide chains connected by a binding protein, is a pigment protein. Hemoglobin (Mw-64 500) is added to excess potassium cyanide (K3Fe(CN) 6, Mw-327.25), and hemoglobin reacts with high-iron potassium cyanide to produce high iron hemoglobin (MetHb). In order to remove excess high-speed iron potassium cyanide, get a purer MetHb sample, the above mixture through the cross-linked glucosaccharide gel column, with phosphoric acid buffer washed away, from different colors, can be directly observed MetHb (reddish-brown) washed away faster, and
small molecules
high-iron potassium cyanide (yellow) washed out slowly, to achieve the goal of completely separating MetHb.II, experimental materials,
reagents
and instruments:
(i) materials: chicken anticoagulant whole blood
(ii) reagents:
1. Carbon tetrachloride (CCl4)
2. Physiological saline
3. Cross-linked glucosamine G-25 (fine grain)
4. 0.1mol/L
phosphate buffer
(pH7.0):
a, 0.1M sodium dihydrophosphate; b, 0.1M sodium phosphate dihydrophosphate;
a, b mixed at a ratio of 49:51, and proofed
5. 0.4% K3Fe (CN)6.
6. Cold physiological saline
(iii) instrument (slightly)
, experimental steps:
1. Treatment of the gel:
take 4 grams of Sephadex-G25 dry powder, soak in distilled water fully soothing (room temperature, 6 hours), then tilt the method to remove the surface suspension of small particles, and finally add the same volume pH7 phosphoric acid buffer to continue soaking.
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