2 Dimensional Gel Electrophoresis
General:
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Always use clean gloves, bench, lab coat.
Weigh using analytical scale.
Use chemical bought only for 2D, pour chemical out rather than using spatula.
Wash all dishes with special soap for 2 D apparatus and rinse with ddh2O.
Degas mineral oil with N2 for 30 minutes.
Solutions:
IEF Buffer I
25 ml
use 50 ml Falcon tube
8 M Urea
2 M ThioUrea
4 % Chaps
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Add ddH2O to 15 ml, dissolve by warming in hand. Add ddH2O to 24.5 ml, add
trace Bromophenol Blue. Add 0.4 g Serdolit MB-1 (SERVA). Incubate 10 minutes
rotating. Filter with Whatman #4 paper. Aliquot 980 ul in screw cap tubes, freeze in
-80 C.
To prepare:
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12 g
3.81 g
1.0 g
Add 10 ul of 1.5 M DTT (fresh 231.4 mg/ml)
Add 1 ul TCEP stock
Add 7.5 ul Pharmalytes IPG buffer
Sample can be precipitated with acetone or TCA, pellet by centrifugation.
Resuspend/Dilute sample in IEF I buffer.
Preparing Strips and Samples:
Prepare sample:
-
-
13 cm strip
290 ul IEF buffer
18 cm strip
400 ul IEF buffer
Prepare IEF buffer with fresh ingredients: 10 ul 1.5 M DTT, 10 ul TCEP, 7.5 ul
Pharmalytes. Add appropriate volume to pellet. Vortex to suspend. Incubate on
bench for 3 hours with sonicating and vortexing.
Spin 27 000 rpm for 30 minutes at RT in ultracentrifuge (Weiner Beckman rotor
100.2).
Take strip out of freezer. Transfer supt to eppendorf. Load volume in strip holder
equally along bottom (freshly cleaned strip holder). Remove plastic cover from IEF
strip. Lay gel side down on sample in strip holder. Make sure to push pointy end to
the pointed side of strip holder. Layer with mineral oil (600 ul for 13cm, 1 ml for
18cm). Lay on IEF machine.
Use program 4 for 13cm 3-10 analytical
Use program x for 18 cm 3-10 analytical. There are other programs specialized for
different strips.
ULTRACENTRIFUGE
Weiner Lab
Has little tubes as well as eppendorf with screw cap that will fit.
TLA
100
100.2
100.3
25 000 g
25 000 rpm
27 000 rpm
25 000 rpm
150 000 g
63 000 rpm
65 000 rpm
60 000 rpm
Agarose Setting Solution
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1% agarose in running buffer with trace bromophenol blue.
Microwave until melted. Let cool slightly. Use to set strip in gel. Use spacer to create
well for MWM in 13 cm medium gel. With 18 cm, dot MWM on filter paper and let
dry. Dip in warm agarose and let harden. Place at end of strip in gel. Pour agarose
around with pipet trying to avoid air bubbles. Let solidify. Run gel.
SDS Equilibration Buffer
50 mM Tris pH 8.8
6 M Urea
30 % glycerol
2% SDS
Bromophenol blue
ddH2O
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10 ml of 1 M stock
72.07 g
69 ml
4g
trace
to 200 ml
Aliquot in 10 ml falcon tubes, freeze at -20 C. Add 100 mg DTT to 10 ml before use.
Preparing strip for gel:
Soak whatman filter strip with running buffer for strip to lay on after taking out of
IEF machine. Set out all tools such as tweezers and syringes. Prepare equilibration
buffer by adding 100 mg DTT fresh to 10 ml. Vortex and bring to RT.
When strip is finished, write down Volt-hours. Remove from stripholder with
tweezers and lay plastic side down on running buffer soaked whatman filter strip.
Slide strip into long glass testube and pour 10 ml prepared equilibration buffer on
top. Cap and put on nutator for exactly 10 minutes!!
Prepare agarose setting solution and MWS. When 10 minutes is over, set up gel in
holder. Remove strip from test tube and dip in running buffer. Load strip onto top
of gel with pointy end facing left. Make sure it is stuck to back plate and push it
down until it is in contact with the gel. Make sure there are no bubbles between gel
and strip. Fill with agarose setting solution with spacer for MWS or filter with MWS.
Make sure there are no bubbles. Run gel at 35 mA per gel or 80 Volts overnight.
SDS-PAGE
Make fresh solutions for SDS-PAGE.
Prepare medium sized gel for 13 cm strip, large gel for 18 cm strip.
13 cm Medium Gel:
2-10% gels
H2O
32 ml
Separating buffer
20 ml
Acrylamide
26.8 ml
Ammonium persulfate 350 ul
Degas for 30 minutes at RT without TEMED.
Add TEMED
30 ul
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Pour gel.
18 cm Large Gel:
12-1.5mm 10 %
Use volumetric flasks to be accurate
Acrylamide
432 ml
1.5 M Tris-Cl separating
325 ml
H2O
513 ml
10% SDS (fresh)
13 ml
10% APS (fresh)
13 ml
degas 30 minutes, RT
add TEMED
110 ul
Displacing solution: 50 ml 1.5 M Tris, pH 8.8
100 ml glycerol
50 ml H2O, trace bromophenol blue
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Clean all glassware and plexiglass stand with soap and rinse with ddH2O. Let dry.
Assemble casting stand:
Sheet : 25mm plexiglass : sheet : 25mm plexiglass : sheet : 12mm plexiglass : sheet :
6mm plexiglass:sheet : glass sandwich : sheet etc…(X12).
Put tube with funnel into casting stand. Fill reservoir with displacing solution. Pour
gel solution through funnel to fill gels to top with gel solution. Remove tube from
reservoir. Let displacing solution fill bottom until gel is 5 mm from top. Layer each
gel with 1 ml isobutanol. Let solidify for one day. Dissemble and wrap each gel
separately. Remember to put paper label in corner of each gel – able to tell them
apart.
Running Buffer for Large Apparatus
20 L ddH2O
60.5 g Tris
288 g Glycine
20 g SDS
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Turn pump on. Pour in chemicals; let mix at RT for 45 minutes. Take out gel
holders, turn down temp to 10 C. Let mix until ready for use.