7 Size Exclusion Chromatography PDF

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7 Size Exclusion Chromatography...

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Size Exclusion Chromatography

I.

Pre-Laboratory. a) What scientific concept(s) will be learned in this lab activity?  The scientific concept of size exclusion chromatography will be learned. This experiment will explain that the small molecules go down the column faster than the bigger proteins that will go slower. b) What laboratory techniques will be learned in this activity?  Gel filtration technique with the separator column will be learned in this experiment. The use of this column is similar to the titration procedure of cleaning it and adding solutions drop by drop. c) What is the objective (or goal) for the lab?  The goal for this lab is to be able to separate proteins depending on their relative size and molecular weight and to be able to explain the principles behind gel exclusion chromatography purification of small molecules.

II.

Materials and Methods. Dr. Pattison’s BCHS 3201 protocol, pages 104-109. The same procedure was followed and there was no change.

III.

Results. Table 1 – Tabulation of Protein Collected From Every Fraction of 1 ml Sample with Measured Wavelength and Specific Color

Fraction

OD550

Comment (color)

1 2 3 4 5 6 7 8 9 10 11 12 13

0.01 0 0 0.01 0.01 0.02 0.11 0.59 0.13 0.05 0.08 0.15 0.20

Clear Clear Clear Clear Clear Clear Light Blue Blue Light Blue Clear Peach Light Orange Light Orange

Fraction

OD550

Comment (color)

14 15 16

0.22 0.17 0.11

Orange Light Orange Peach

17 18 19 20 21 22 23 24 25 26 27 28

0.07 0.04 0.02 0.01 0.01 0 0 0 0 0 0 0

Peach/Clear Light Yellow Yellow Yellow Yellow Light Yellow Light Yellow/Clear Clear Clear Clear Clear Clear

Graph 1 – Absorption Versus Fraction Number of 1 mL When the Wavelength is 550 nm

Absorabnce (χ=550 nm) Vs. Fraction Number (1 mL) 0.7

Blue Dextran

Absorption (550 nm)

0.6 0.5 0.4

BSA Cytochrome c

0.3

Potassium Chromate

0.2 0.1 0 0

5

10

15

20

25

Fraction (mL)

Table 2 – Tabulation of the Kav Values Calculated Versus the Molecular Weight of Every Single Protein Extracted From the Samples Sample Log MW Kav Blue Dextran BSA Cytochrome C Potassium Chromate

6.301 4.819 4.14 2.288

0.111 0.389 0.444 0.667

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Graph 2 – Plot of the Kav Values Obtained from the Formula Versus the Proteins that are Identified by Their Molecular Weight

Kav Vs. Log of Molecular Weight of Proteins 0.8

Cytochrome C

0.7

BSA

0.6 0.5

Potassium Chromate

0.4 0.3 0.2

Blue Dextran

0.1 0 2

IV.

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

Discussion/Post-Lab Questions. Calculations: Vo = void volume = the volume outside the beads (before anything is eluted) = 6 mL Ve = elution volume for the sample (highest peak)  For blue dextran, Ve = 8 mL  For BSA, Ve = 13 mL  For cytochrome c, Ve = 14 mL  For potassium chromate, V e = 18 mL Vt = the bed volume (clear color after everything is eluted) = 24 mL Formula: (Ve −Vo ) Kav= (Vt −Vo) Sample calculation for blue dextran: (8−6) = 0.111 Kav= (24−6) Post Lab Questions: 1. Create a graph of absorbance versus fraction number at 550 nm. Label the identity of the peaks.  It is shown in the results section and labeled as graph 1. 2. What is the void volume (Vo) for your column?



Vo is the volume outside the beads before anything starts eluting in the column. It is the last data when the color was clear before the light blue started to merge. In this experiment, it was 6 mL. 3. What is the bed volume (Vt) for your column?  It is the bed volume after everything has eluted outside the column. It is the first clear color after the yellow potassium chromate elutes completely. The wavelength should be 0 nm for it. In this experiment, it was 24 mL. 4. What is the Ve for the blue dextran, cytochrome c, and the potassium chromate in the sample? How would you figure out this value for BSA?  It is 8 mL for blue dextran, 14 mL for cytochrome c, and 18 mL for potassium chromate. For BSA, it is one of those proteins with no color that have to be seen under wavelength of 280 nm instead of 550 nm like other proteins. Then the wavelength can be recorded as OD280 and the concentration of the protein in the fractions can be found from the Bradford Assay. 5. Determine the Kav for the blue dextran, cytochrome c, and potassium chromate in the sample. (Ve −Vo )  Formula: Kav= (Vt −Vo) (8−6) = 0.111 (24−6) (14−6) = 0.444 Cytochrome c  Kav= (24−6) ( 11−6) = 0.389 BSA  Kav= (24−6) (18−6) = 0.667 Potassium chromate  Kav= (24−6) 6. Plot the Kav vs Log molecular weight for blue dextran, cytochrome c, and potassium chromate. You know the molecular weight of BSA. Use the graph to estimate the K av of BSA.  It is all shown in the Results section and labeled as Graph 2.

Blue dextran  Kav=...