Transcript Gel filtration chromatography

BIOCHEMISTRY EXPERIMENT
Principle + Practice
Logic analysis
 Professor: Sheng Zhao (赵晟)
 Web for lecture slides：
http://teaching.ewindup.info
 Email/QQ /MSN/gchat:
[email protected] or [email protected]
 Mobile:18551669724 or 18761413925
Class syllabus by logic
 Protein quantification: Folin phenol (Lowry) assay
 Ion exchange chromatography for the mixed amino
acids
Protein analysis
 Transamination
 Protein purification
 Gel filtration chromatography
 Sodium dodecyl sulfate-Polyacrylamide gel
electrophoresis (SDS-PAGE)
Lipid analysis
Nucleic acid analysis
 Serum triglyceride (TG) measurement
 Plasmid DNA extraction, restriction enzyme
digestion, and agarose gel electrophoresis
 Enzyme Km
Protein function
 Serum glutamic-pyruvic transaminase (ALT)
measurement (Exam!)
Class syllabus by real experiments
1. Introduction and Protein quantification: Folin phenol (Lowry) assay
2. Protein purification: Gel filtration chromatography
3. Plasmid DNA extraction, restriction enzyme digestion, and agarose
gel electrophoresis
4. Enzyme Km
5. Transamination
6. Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDSPAGE)
7. Ion exchange chromatography for the mixed amino acids
8. Serum triglyceride (TG) measurement
9. Serum glutamic-pyruvic transaminase (ALT) measurement (Exam!)
Experiment: isolation of hemoglobin using the
gel filtration chromatography
Charge, size, solubility, and Specific binding, etc.
Column chromatography
• Charge
• Ion-exchange chromatography:
• Isoelectric focusing chromatography
• Size
• Size-exclusion chromatography
• Solubility
• Hydrophobic interaction chromatography
• Specific binding
• Affinity chromatography
Column chromatography
 Size-exclusion
Chromatography
 This method separates proteins according to their size.
 The column contains a cross-linked polymer with pores of
selected size.
Size Exclusion Chromatography:
Applications of gel filtration
• Purification of enzymes and other proteins.
• Estimation of molecular weight mainly for
globular proteins
• Desalting
• The method of gel
filtration
chromatography can be
used to separate
hemoglobin (Hb) from
riboflavin according to
their molecular weight.
• Hb (MW: 64500 ):
• Riboflavin (MW: 376)
Regents:
1. Sephadex
G-50;
2. 0.9 % NaCl
3. Non-coagulating blood
4. Riboflavin
G50
Equipment and apparatus:
1.
Chromatography column
(diameter, 0.8-1.5 cm; length,
17-20 cm);
2.
Bracket for chromatography
column;
3.
Centrifuge tube, burette,
centrifuge.
1. Column filling
1. Sephadex G-50 will be used for the column filling
2. While filling the column, please notice that the
chromatography column fixed on the bracket should be
upright vertically.
3. Close the clamp for exit at the bottom, and add gradually
and gently the sephadex G-50 suspending solution from
the top of column.
4. When approximately 1-2 cm volume of sephadex G-50 is
already deposited in the column, open the clamp for exit
and the outflow speed should be adjusted as 1 ml/min.
5. Continue to finish the filling sephadex G-50 suspending
solution gradually without drying the column.
NOTICE:
1. NO bubble and layered deposition
2. Flat column surface (or the formed color band will be
irregular)
3. Tightly closed clamp for exit
4. Never dry the column, and approximately 0.5 cm elution
solution should be retained on the top of the surface of
sephadex G-50 resin.
2. Sample preparation for hemoglobin
1.
Add 1 ml blood into the centrifuge tube, balanced, and then centrifuge
for 5 min at 3000 rpm.
2.
Discard the supernatant (blood plasma), add 10 ml 0.9% NaCl, and mix
thoroughly of the NaCl with blood corpuscle. Centrifuge again as above
3.
Discard the supernatant and add 10 ml 0.9% NaCl again to wash the
blood corpuscle for one more time.
4.
And then, dilute the blood corpuscle with five volume of ddH2O (Why?).
5.
Before the gel filtration, mix three drops of blood preparation with three
drops of Riboflavin solution.
Preparation of hb from blood:
3. Samples loading
1. Slightly open the clamp for exit to make the solution in column outflow
slowly till the surface of sephadex G-50 resin just appears from the solution,
then close tightly the clamp for exit. Do not make the surface of resin
become dry, or your experiment will be failed.
2. Add the mix sample with burette on the surface of sephadex G-50 resin
gradually along the inner wall, and do not destroy the surface of sephadex
G-50 resin.
3. Open the exit to make the sample run into the stationary phase. While the
surface of sephadex G-50 resin appears again, add quickly the 0.9% NaCl
to elute the sample with the elution speed of 1ml/min.
4. Observe the elution and separation of Hb and riboflavin.
4. Recording the data
• Record the elution sequence of the color.
• Notice: the band color for hemoglobin is red, and the band
color for riboflavin is yellow.