Resolution of Proteins: SDS-PAGE
Cast Resolving Gel
1.
2.
3.
4.
5.
Clamp glass “gel-casting” plates into plastic “gel-casting” tray (found in marked drawers)
 Use 1.5mm plates for ~50µl of sample, 0.75mm for less
Fill space between glass plates with water, watch to make sure no leaks
 If leaks, re-position, re-clamp, and test again with water
Make Resolving Gel in small beaker. Higher % = higher degree of separation
Pour out water from between glass plates. Pour in Resolving Gel up to bottom of green bar
 Top off with methanol or water
Clear distinction between water and gel indicates gel had dried (~20 minutes). Proceed with stacking gel.
Cast Stacking Gel
1.
2.
3.
Make 4% Stacking Gel in small beaker. Higher % = higher degree of separation
Pour off water from separating gel. Pour in stacking gel stacking gel up to very top
Place clean comb in gel, make sure 1.5mm comb for 1.5mm plate, etc.
Prepare/Denature Protein Samples for Separation: From BL21 expression clones (Bradford not necessary?)
1.
2.
3.
4.
Re-suspend pellet in appropriate volume of Sample Buffer to lyse cells, and denature (linearize) proteins
 Ex. Add equivalent volume of 2X Sample buffer to pellet
 Ex. Add 75 µl Sample buffer and 75 µl PBS to pellet
Use 22 guage needle to shear sample by repeatedly withdrawing and depositing in epindorff tube
 Pulse spin to spin down any froth
Secure epindorff with guard to prevent tube from popping open
Boil sample 5 minutes at 95°C in heating block
Assemble SDS-PAGE Apparatus and Resolve protein Samples
1.
2.
3.
4.
5.
Lock glass plates (with solidified gel) into gel electrophoresis cassette
 Short plate faces inward; use spacer with “short plate” inward if only separating 1 gel
Place gel electrophoresis cassette into SDS-PAGE apparatus
Fill inner chamber with running buffer and outer chamber half way if inner chamber doesn’t leak
 If inner chamber leaks, fill all the way
Carefully remove comb, load ladder (BioRad) [-20 SDS-PAGE BOX], load 10-50 µl of sample into wells
Electrophorese at 100V ~1-1.5 hrs
Bradford Assay: Determine amount of protein in sample and volume to load in gel
1.
2.
3.
4.
5.
6.
7.
8.
9.
Turn spectrophotometer on 15 minutes before measurement (Press VIS)
Prepare control cuvette: 98µl 0.15M NaCl + 1ml PDR (Bradford Reagent) + 2µl MP40 lysis buffer
Prepare sample cuvette: 98µl 0.15M NaCl + 1ml PDR (Bradford Reagent) + 2µl cell lysate
Set wavelength to 595 nm (Press “595” “Enter” “Lambda”)
Place control cuvette in holder (clear side facing reader). Close lid, Press CAL.
 Record OD; Reading ≈ 0 ABS. (Press ABS if set to %)
Replace control with sample cuvette. Obtain 2 readings (both ~ consistent)
Once finished with spectrophotometer, remove cuvette, press VIS
Open up “Standard Curve” Excel file. Input sample values for “OD-1” and “OD-2”
Algorithm calculates volume of cell lysate required to load for specific amount of protein
 Ex. For CaM Binding Assay, want to load 0.5 mg protein/ml. Use C1V1=C2V2
o C1 = (conc. from Bradford assay)
o C2 = 0.5 mg / ml
o V2 = 1 ml
o V1 = ?
Example:
OD avg
0.7005
ug/ul
4.050877
V (µl)
7.405803
x2 (µl)
14.81161
Desired Amount of Total Protein
30ug
µg/ml = concentration of total protein in this sample of cell lysate
V = volume of sample which contains specified amount of protein (30ug)
x2 = vol. of sample to load (after diluting entire sample 1:1 with 2X SDS) so total of 30ug is loaded
 Total Volume = (½ Cell Lysate) + ( ½ 2X SDS)
 14.81161 µl = (7.405803 µl Cell Lysate) + (7.405803 µl 2X SDS)
x1.33 = vol. of sample to load (after diluting 3:1 with 4X SDS) so total of 30ug is loaded
 Total Volume = (¾ Cell lysate) + ( ¼ 4X SDS)
For 4X SBS, dilute 4X SBS 1:1 (with ddH2O) to make 2X SBS, then dilute that solution 1:1 with lysate. This gives
solution equivalent to 1:1 dilution with 2X SDS, so load same amount
 Total Volume = (½ Cell Lysate) + ( ¼ 4X SDS) + (¼ ddH2O)
 14.81161 µl = (7.405803µl Cell Lysate) + (3.7029015µl 4X SDS) + (3.7029015µl ddH2O)
Visualize Separated Proteins: Coomassie Stain
Stain Gel With Commassie Dye
1.
2.
3.
4.
5.
6.
Disassemble resolving cassette and pry open plates slowly so gel falls on short plate (protein side up)
Use wedge to cut off stacking gel and cut top corner of gel above ladder lane for orientation
Deposit gel in Tupperware, submerge in Coomassie stain, incubate shaking at RT for 2 hours?
Drain coomassie stain, replace with Destain Solution, incubate shaking at RT O/N.
Replace destain solution when possible upon color saturation
“Quick and Dirty Destain”: Submerge stained gel in water, microwave 3 minutes, replace water, repeat X3.
Visualize Separated Proteins: Western Blot
Transfer Resolved Proteins from Gel to Membrane
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Fill large tub with ice, obtain large glass bowl (different sizes for 1 vs 2 gels), and small Tupperware
Place 2 black transfer sponges (rinsed with water) in glass bowl
Use glass plate to cut 2 pieces of filter paper from large role [on floor beside centrifuges]
Place filter paper pieces on black sponges in bowl, immerse in transfer buffer
Change gloves, obtain role of nitrocellulose membrane [on floor beside deli fridge]. Cut piece size of gel.
 Maybe make tracer? Use gel plate?
IMPORTANT: NEVER TOUCH WHITE MEMBRANE. Only touch blue paper around edges of cut piece
 Recommended to use tweezers to pick up piece at edge to minimize contact
Cut top left corner of membrane above ladder lane for orientation
Put membrane in Tupperware filled with water. Let sit for 2-3 minutes to activate membrane
Pour out running buffer from SDS-PAGE apparatus, remove resolving cassette, wash out apparatus well
Disassemble resolving cassette and pry open plates slowly so gel falls on short plate (protein side up)
Use wedge to cut off stacking gel and cut top corner of gel above ladder lane for orientation
Place gel on filter paper (which is on sponge). Place sponge on black face of cassette (protein side up)
 For each layer of sandwich, flatten with wedge soaked in transfer buffer to remove bubbles
Place membrane on top of gel (contacts protein side), and place nitrocellulose and other sponge on top
Close cassette, fit into electrophoresis cassette with black on black, and clear face facing red
Insert cassette into SDS-PAGE apparatus (match lead colours), push down all the way until clicks
 Fit dummy ice pack [in deli fridge?] in other space if transferring only 1 gel
Fill apparatus with transfer buffer half way if only 1 gel (full if 2 gels), and place in tub of ice
Double check leads match colours when securing lid. Electrophorese 1 hour at 100V.
Hybridize Primary Antibody
1.
2.
3.
4.
5.
Block membrane in ~20ml of 3-5% Milk TBST at least 1hour shaking in Tupperware at RT
Prepare antibody dilution in blocker (prepared milk TBST)
After block, pour out Milk-TBST and replace with diluted 1° Ab. Shake O/N at RT in Tupperware.
Dilute 2° Ab (from animal in which hybridoma produced), hybridize as above
 Usually mouse 2° Ab dilution = 1 : 10,000 Ab: block