Laboratory No. 5:
Purification of Green & Blue Fluorescent Proteins (Edvotek #108)
By: Kazi M. Rahman
Instructor: Stephen Brown, Ph.D.
Los Angeles Mission College
Biology 006, Section: 0142
March 3rd, 2011
Lab Partners:
Lucia Sandoval
Nicole Park
I.
Introduction
The purpose of this lab is to purify the green (gfp) and blue (bfp) fluorescent
proteins using gel chromatography and to accurately estimate the protein
polypeptide size by analyzing the fractions of gfp and bfp by denaturing
polyacrylamide gel electrophoresis.
The GFP and BFP proteins are known as chimeric proteins. Chimeric
proteins, also known as fusion proteins, are created through the joining of two or
more genes. It is helpful in purification process as they can be detected using
long or short ultraviolet light based on their fluorescence. The amino acids SERTYR-GLY in positions 65-67 in the amino acid sequence of GFP accounts for the
color of the GFP, whereas, BFP is a derivative variant of GFP and the HIS-66
amino acid is substituted at the TYR-66 position of the GFP and TYR-145 to PHE145. We will pour the samples of GFP and BFP through the gel filtration matrix
and collect the fractions after it reaches the bottom.
We will identify, using long wave U.V. light, the tube with the highest
fluorescence of gfp and bfp. The samples will then be denatured for molecular
weight analysis. Proteins are denatured by adding denaturing solutions
containing SDS and 2-mercaptoethanol and boiling for 5 minutes. Proteins lose
their folding and tertiary structure of R-group interactions by boiling in the
presence of SDS and 2-mercaptoethanol. Denatured standard protein markers of
known molecular weights and our gfp and bfp are separated by electrophoresis
in parallel and will be used to estimate molecular weights of the unknowns by
graphical analysis.
II.
Materials and Methods
a. Materials
i. Matrix
ii. Chromatography Column
iii. Elution Buffer
iv. 1 ml pipet and pipet pumps
v. Transfer pipet
vi. 250 ml Beaker
vii. 16 micro test tubes
viii. Cell extract containing green protein (gfp)
ix. Cell extract containing blue protein (bfp)
x. Protein molecular weight standards
xi. Protein denaturing solution
xii. Vertical gel electrophoresis apparatus
xiii. Long wave U.V. lamp
xiv. Polyacrylamide gel
xv. Glacial acetic acid
xvi. Methanol
b. Methods
i. Packing the Column
1. Mix the matrix completely by swirling or gently stirring.
2. Pour the slurry into the column.
3. Using a transfer pipet, add elution buffer to fill the reservoir.
4. Place an empty beaker under the column.
5. Let the buffer flow through the column for approximately 10
minutes and the matrix will pack down into the column
ii. Fraction Collection
Collecting Column Fractions of (gfp) Protein
1. Label 8 test tubes 1-8.
2. Pour 0.2 ml gfp extract onto the top of the matrix bed and let
the sample go down the walls of the column.
3. Place a beaker under the column
4. Add a few drops of buffer over the bed with a transfer pipet
5. Keep adding buffer little bit at a time
6. Collect 0.5 mL fractions in each of the test tubes labeled 1-8
7. Check all fractions using long wave U.V. light to identify tubes
that contain the gfp protein.
Collecting Column Fractions of (bfp) Protein
8. Wash the column with 10ml of 1x elution buffer
9. Label a second set of 8 test tubes 9-16.
10. Pour 0.2 ml of bfp protein onto the top of the matrix bed and
let the sample go down the walls of the column
11. Repeat steps 3-7.
Sample Preparation for Denaturing SDS-Gel Electrophoresis
12. Identify tube with highest fluorescence of the gfp and bfp
transfer 200 µl into four clean test tubes labeled gfp native, gfp
denatured, bfp native and bfp denatured.
13. Add 25 µl of protein denaturing solutions to each tube labeled
gfp denatured and bfp denatured.
14. Boil for 5 minutes and allow to cool to room temperature
Electrophoresis of proteins
15. Load the standard protein markers and gfp and bfp native and
denatured sample onto the electrophoresis gel
16. Run the gel
17. After electrophoresis, slide the gel into a staining tray and stain
with Protein InstaStain® with the stain side (blue) in the
liquid.
18. After staining, Proteins bands will appear medium to dark blue
against a light blue background and will be ready for graphical
analysis.
III.
Results
a. GFP colors observed after gel filtration
Tube 1
b. B
F
P
Tube 2
Bright
Darkest
Green
green
Tube 3
Tube 4
Tube 5
Tube 6
Tube 7
Bright
Clear
Clear
Clear
Clear
Tube 8
clear
b. colors observed using long wave U.V. light after gel filtration
Tube 9
Tube 10
Tube 11
Tube 12
Tube 13
Tube 14
Tube 15
Tube 16
Bright blue
Darkest
Light blue
Clear
Clear
Clear
Clear
Clear
blue
C. Electrophoresis analysis
Table of Migration distance of standard protein markers and GFP denatured
Migration Distance (cm)
Molecular Weight (Daltons)
1.7
94,000
2.3
67,000
3.1
38,000
4.7
30,000
6.0
20,000
4.9
Denatured GFP and BFP
IV.
Conclusion
Our tubes #2 had the highest fluorescence of the gfp and our tube #10 had
the highest fluorescence of bfp under long wave U.V. light. That means we were able
to capture the purified gfp and bfp sample from the cell extract. According to the
graphical analysis of the electrophoresis table, our molecular weight estimation is
24,000 Daltons for the gfp and the bfp proteins. The actual molecular weight of the
gfp and bfp protein is 40,000 Daltons. Our graphical analysis shows a 40% error in
the experiment.
Many errors may have contributed to such high percent error. First, an
incorrect gel concentration can result in samples running too quickly. Gel can also
be compromised if the gel has cuts or bubbles while loading the samples. Even a
small percentage can affect the results. Although it seems simple enough, loading
samples into the small wells in the gel is difficult. Too much of the sample could
cause it to smear or appear smaller than it truly is; too little of the sample may be
impossible to see. Problems with visualization method while measuring can also
hinder results. While measuring the length of gel traveled with ruler, parallax can
lead to poor accuracy of measurement.
Overall, we have gained quite a bit of experience purifying and determining
the size of Green (gfp) and Blue (bfp) Fluorescent Proteins. Such, knowledge of
techniques and possible errors, can help us in future research endeavors.