UBHD Student Laboratory Procedure:
Willy's two most pressing issues are to (1) separate the newly identified contaminant
from the UltraHair protein additive, and (2) incorporate the separation technique and
assay for purity of the UltraHair protein additive in the SOP QC assay. As a research
associate, you will take the 0298/12 batch of protein additive and determine if the
contaminant can be separated from the desired UltraHair additive on two different
chromatography resins. You will analyze the separation obtained on the two columns
using SDS-PAGE.
Index of Procedures:
I.
G50 Molecular Sizing Column
p.2
II.
SP-Sepharose Ion Exchange Column
p.4
III.
SDS-PAGE Analysis of Column Fractions
p.6
IV.
Solutions used for columns and SDS-PAGE
p.9
1
I.
G50 Molecular Sizing Column:
Materials Needed:
5ml plastic disposable pipette with glass wool, tygon tubing and pinch clamp (column)
Ring Stand and clamp (to hold column)
Pasteur pipettes and bulb
1.5ml microfuge tubes and rack
0.1M potassium phosphate buffer, pH 7.0
G50 resin (8ml volume of settled resin/column, dry resin swollen in 0.1M potassium
phosphate buffer, pH 7.0 overnight at 40C)
Small beaker for waste collection
250 l Batch 0298/12 UltraHair Protein Additive (protein mixture), hold on ice
Sharpie pen
Safety glasses
Disposable gloves
Procedure:
1.
Collect all materials at your lab bench.
Set up ring stand and clamp. Place column in vertical position. Place your
discard beaker under the column with column tubing inside the beaker.
Label 8 microfuge tubes (G1-G8). Mark a 1ml volume on each tube.
2.
Make sure the column is securely clamped off. Add 3-4ml of 0.1M potassium
phosphate buffer, pH 7.0 to column.
3.
Slowly swirl G50 until the resin is well mixed. Using a Pasteur pipette, add the
resin slurry slowly, but in a continuous stream, to the column and carefully open
the column. (You want to fill the column with 8ml of packed resin that has been
added in a continuous flow without either overflowing or letting your column run
dry. The resin slurry packs as the buffer flows through.) Do not allow the column
to run dry! Add additional buffer as needed to keep the buffer level above the
packing bed of resin. If you see air bubbles in the column, gently tap the column
to dislodge. Clamp off column securely when resin has settled, leaving a buffer
layer on top of the bed.
4.
The finished column should have an 8ml settled resin bed (pipette will be filled to
lower blue line.) Wash the column with 8ml of 0.1M potassium phosphate buffer,
pH 7.0 (one column volume). Allow the fluid level to just reach the top of the
resin bed and clamp the column off.
5.
Carefully layer 100l of your protein mixture on top of the resin bed. Do not
disturb the resin bed! Unclamp the column just long enough to allow the 100l
of your protein mixture to enter the resin. Immediately clamp off the column.
6.
Carefully layer 100l of 0.1M potassium phosphate buffer, pH 7.0 on top of the
resin bed. Unclamp the column just long enough to allow the 100l of buffer to
enter the resin. Immediately clamp off the column. Repeat with another100l of
buffer. (This 200l of buffer washes the protein mixture into the column.)
(Save extra protein mixture for ion exchange column and to run on gel)
2
7.
Carefully layer 0.1M potassium phosphate buffer, pH 7.0 onto the column.
Continue to add buffer as you unclamp the column and begin collecting 8-1ml
fractions in the labeled microfuge tubes. When all 8 fractions are collected, clamp
off the column. Note any color differences in the collected fractions. Save the
fractions. Label G50 column fractions. Hold on ice; freeze at -200C until fractions
are run on an SDS-PAGE gel.
Color of collected fractions:
G1
_____________________
G2
_____________________
G3
_____________________
G4
_____________________
G5
G6
G7
G8
_____________________
_____________________
_____________________
_____________________
Remove cotton plug and
discard
To Make the G50
Column
QuickTime™ and a
TIF F (Uncompressed) decompressor
are needed to see this picture.
Approximate fill line to obtain an 8
ml packed bed volume of G50 resin
3
II. SP-Sepharose Ion Exchange Column:
Materials Needed:
Poly-prep chromatography column (Bio-Rad)
Ring stand and clamp (to hold column)
Pasteur pipettes and bulb
1.5ml microfuge tubes and rack
0.1M potassium phosphate buffer, pH 7.0
1.0M KCl, 0.1M potassium phosphate buffer, pH 7.0
SP-Sepharose (1.5ml volume of settled resin/column)
Small beaker for waste collection
250 l Batch 0298/12 UltraHair Protein Additive (protein mixture), hold on ice
Sharpie pen
Safety glasses
Disposable gloves
Procedure:
1.
Collect all materials at your lab bench.
Set up ring stand and clamp. Mark 1.5ml on the side of the column. Place column
in vertical position. (Tubing may be added to column to help control flow rate of
column.)
Label 6 microfuge tubes (W1-W3 and E1-E3). Mark a 1ml volume on each tube.
2.
Make sure the column is stoppered or clamped off. Add 1-2ml of 0.1M potassium
phosphate buffer, pH 7.0.
3.
Swirl the SP-Sepharose until the resin is well mixed. (Have extra buffer ready to add to
the column with a Pasteur pipette.) Pour slurry into the column and open the column. Do
not allow the column to run dry! Add buffer to keep the buffer level above the resin
bed. If you see air bubbles in the column, gently tap the column to dislodge.
4.
The finished column should have a 1.5ml settled resin bed. Wash the column with 3ml of
0.1M potassium phosphate buffer, pH 7.0 (2 column volumes). Allow the fluid level to
just reach the top of the resin be and stopper or clamp off the column.
5.
Carefully layer 100l of your protein mixture on top of the resin bed. Do not disturb the
resin bed! Unstopper or unclamp the column just long enough to allow the 100l of your
protein mixture to enter the resin. Immediately stopper or clamp off the column. (Save
extra protein mixture to run on gel)
6.
Carefully layer 100l of 0.1M potassium phosphate buffer, pH 7.0 on top of the resin
bed. Unstopper or unclamp the column just long enough to allow the 100l of buffer to
enter the resin. Immediately stopper or clamp off the column. Repeat with another100l
of buffer. (This 200l of buffer washes the protein mixture into the column.)
7.
Carefully layer 3ml of 0.1M potassium phosphate buffer, pH 7.0 onto the column.
Unstopper or unclamp and collect 3-1ml fractions (W1-W3) in the labeled tubes. (Keep
track of the buffer level in the column as you collect, and don't let the level drop below
the resin bed. You may need to add a few extra drops of buffer.) Stopper or clamp off the
column. Hold fractions on ice.
4
8.
Carefully layer 3mll of 1.0M KCL, 0.1M potassium phosphate buffer, pH 7.0 onto the
column. Unstopper or unclamp and collect 3-1ml fractions (E1-E3) in the labeled tubes.
(Keep track of the buffer level in the column as you collect, and don't let the level drop
below the resin bed.) Stopper or clamp off the column.
9.
Save the fractions, and label SP-Sepharose column. Hold on ice; freeze at -200C until
fractions are run on an SDS-PAGE gel. Note any color differences in the collected
fractions.
Color of collected fractions:
W1 ____________________
E1
____________________
W2 ____________________
E2
____________________
W3 ____________________
E3
____________________
BioRad Poly Prep Column used for SP-Sepharose Ion Exchange Column
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
5
III. SDS-PAGE Analysis of Column Fractions
CAUTION: Although the gels used in this procedure are pre-formed, unpolymerized acrylamide
is a neurotoxin. ALWAYS wear gloves when using these reagents or handling gels. A
polyacrylamide gel is not toxic unless it contains unpolymerized monomer.
Materials:
Mini-Protean II electrophoresis equipment (BioRad) (or similar electrophoresis equipment)
4-20% preformed polyacrylamide gels, 10 well combs (can use either 1 gel/team or 1 gel/2
teams)
Power supply
Micropipette and tips (*gel-loading tips may be used)
Microfuge tubes and rack
Pasteur pipettes and bulb
SDS sample buffer
SDS-PAGE Running Buffer
Coomassie Blue Stain and Destain
Gel Drying Kit (Frame, Cellophane Sheets and soaking solutions) or Gel Documentation System
Boiling Water bath or Heat Block
Single-edge razor blade
Safety glasses
Disposable gloves
Fractions from both the G50 and SP-Sepharose Columns, Protein mixture (Batch 0298/12) (Keep
on ice)
Molecular Weight Standards (NOVEX SeeBlue Pre-stained Standards or similar purchased set)
(Keep on ice)
Procedure:
1.
Assemble materials and samples to be run on gels.
2.
Remove the gel cassette from its leak proof pouch
3.
Remove the comb by positioning your thumbs on the ridges located on each side of the
comb and pushing upward with a smooth, continuous motion.
4.
Set the gel cassette down on a flat surface with the tape with the black line facing up.
With the razor blade, cut the tape along the black line. Pull the tab across the gel to
remove the bottom strip of tape. Note: the strip must be removed from the bottom of the
cassette before the gel will make contact with the buffer in the lower buffer tank. If you
forget to do this, your proteins won't move or will move VERY slowly)
5.
Place the gel into the Mini-Protean II cell clamp assembly, aligning the gel and clamp on
a flat surface. Finger-tighten the 4 screws on the clamp assembly. Attach the gel
sandwich to the inner core of the Mini-Protean II cell. Place unit in the gel box, and fill
upper and lower chambers with Running Buffer (~300ml total). With a Pasteur pipette,
rinse the wells with running buffer, and straighten any wells which may have been
distorted during comb removal and rinsing. Note: If the level of the buffer in the upper
chamber goes down, the unit is leaking. You can try and tighten the screws to stop the
leak, or, add enough buffer in the outer chamber to equalize the levels (this takes much
more buffer).
6
6.
If using 1 gel / team:
Label 10 microfuge tubes as follows:
Label
Amount of Sample Amount SDSsample buffer
MW standards
10 l
L (protein mixture)
2 l
2 l
G1-G2
20l
20l
G3
20l
20l
G4
20l
20l
G5
20l
20l
G6
20l
20l
G7-G8
20l
20l
W1-W3
20l
20l
E1-E3
20l
20l
Amount loaded
onto gel
10 l
4l
15l
15l
15l
15l
15l
15l
15l
15l
If using 1 gel / 2 teams:
Label 10 microfuge tubes as follows:
Label
Amount of Sample Amount SDSsample buffer
MW standards
10 l
L (protein mixture)
2 l
2 l
Team A: G3-G4
20l
20l
Team A: G5-G6
20l
20l
Team A: Sp-S W
20l
20l
Team A: Sp-S E
20l
20l
Team B: G3-G4
20l
20l
Team B: G5-G6
20l
20l
Team B: Sp-S W
20l
20l
Team B: Sp-S E
20l
20l
Amount loaded
onto gel
10 l
4l
15l
15l
15l
15l
15l
15l
15l
15l
7.
8.
9.
10.
Add SDS-Sample Buffer to the labeled tubes. Next mix the tubes you are going to take
samples from, and then transfer the appropriate amount to the new microfuge tube. (For
example: Mix G1 tube, then transfer 10l to G1-G2 tube. Mix G2 tube, then transfer 10l
to G1-G2 tube, and mix to obtain the needed total of 20l.)
Pole a hole in the top of each tube, and place in boiling water bath or heat block (1000C)
for 1-2 min. Note: You can boil all of your liquid away if you are not careful. If this
happens, you can add additional SDS-sample buffer to reconstitute the proteins.
Load the designated amount (see tables above) onto the gel. Note: This is easier if you
have gel-loading micropipette tips, but regular tips will also work.
Connect the gel box to the power supply. Run the gel at 150 V until the bromphenol blue
tracking dye reaches the bottom of the gel. (~45-50 min)
Turn the power supply off and disconnect the leads. Then disassemble the gel apparatus
and place the gel in the Coomassie blue staining solution and microwave* for 30-45 sec.
Stain for an additional 15 min with agitation. Transfer the gel to destain and microwave*
7
for 30-45 sec. Destain for 30 min-1hr (you can place a Kimwipe to absorb stain and
further decrease the destaining time). Once the gel is sufficiently destained (fairly clear
background and clear bands), proceed to drying the gel or placing it into a gel
documentation system.
*If a microwave is not available, the solutions can be heated to almost boiling before use. It isn't
necessary to heat at all, but this decreases the staining and destaining times. If no heat is used,
the gel should be stained overnight with agitation, and destained with agitation until the
background of the gel is fairly clear and the bands are clear.
BioRad Mini Protean II Unit
Insert that holds cassettes
Cassettes that hold gels
Buffer reservoir
Insert with cassettes in buffer reservoir
Images courtesy of Marcia Bhide (www.edu-graphics.com)
8
IV. Solutions Needed for Columns and SDS-PAGE
Buffer Preparation for Column Chromatography:
1.
0.1M Potassium Phosphate Buffer, pH 7.0
Potassium phosphate dibasic (K2HPO4) FW 174.18
Potassium phosphate monobasic (KH2PO4) FW 136.1
Prepare 0.1M solutions of both dibasic and monbasic potassium phosphate
[M = g/ (MW)(L)]
To make a total volume
of:
100 ml
500 ml
Dibasic solution
Monobasic solution
61 ml
39 ml
306 ml
194 ml
Check the pH of the buffer solution and adjust to pH 7.0 using additional monobasic or
dibasic solution as needed.
2.
1M KCl in 0.1M Potassium Phosphate Buffer, pH 7.0
Potassium chloride (KCl) FW 74.56
To prepare 100ml:
Weigh out 7.5 g KCl and dissolve in ~90 ml of 0.1M Potassium Phosphate
Buffer, pH 7.0. When KCL is in solution, bring total volume to 100 ml.
Solution Preparation for SDS-PAGE:
1.
Sample Buffer (125mM Tris-HCl, 4% SDS, 20% glycerol, 10% -mercaptoethanol)
0.5M Tris-HCl, pH 6.8
2.5 ml
10% SDS
4.0 ml
Glycerol
2.0 ml
-mercaptoethanol
1.0 ml
0.04% Bromphenol blue
0.5 ml
Bring total volume to 10 ml with distilled H2O
Note: Sample buffer can be purchased. Just prior to use -mercaptoethanol is added.
2.
Running Buffer (25mM Tris, 192mM glycine, 0.1% SDS, pH 8.3)
Trizma-base
3.0 g
Glycine
14.4 g
10% SDS
10.0 ml
Bring total volume to 1 L with distilled H2O
3.
Coomassie Blue Stain Solution
Coomassie Blue R-250
2.5 g
9
Methanol
400 ml
Acetic Acid
100 ml
Bring total volume to 1 L with distilled H2O
4.
Destain Solution
Methanol
400 ml
Acetic Acid
100 ml
Bring total volume to 1 L with distilled H2O
10