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Total Protein Isolation by Dry Ice-Ethanol Bath Freeze/Thawing
Dry Ice / Ethanol baths are used to rapidly cool solutions to below freezing temperatures. Dry Ice / Ethanol baths can be used to freeze tissue sections for histology or to rapidly cool Ethanol solutions to induce the precipitation of RNA, DNA, or proteins.
Required:
Ethanol
Dry Ice
Ethanol Solution:
Prepared in ddH2O
70% (v/v) Ethanol*
See Hint #4
1. Find a suitable container to hold the bath (see Hint #1).2. Break the Dry Ice into easily transferable pieces (see Hint #2).
3. Add several pieces of Dry Ice to the bath.
4. Carefully add the Ethanol Solution to the Dry Ice until it covers the Dry Ice in the bath (see Hint #3).
5. After the Ethanol has cooled down, the solution will "boil" more slowly. As the "boiling" slows, add more Ethanol and / or Dry Ice as necessary.
1. The container is usually made of stainless steel or a rubber ice bucket and not plastic. Plastics can crack, causing super cooled Ethanol to leak. Although some high performance plastics can be used as a bath, the plastic will eventually degrade and crack. Find a container with a depth that is appropriate for your application.
2. Be sure to use gloves when handling Dry Ice.
3. Be careful, the Ethanol may splatter and burn you.
4. For general applications, use 70% (v/v) Ethanol, although other concentrations of Ethanol can be used. Remember that as you lower the concentration of Ethanol in the bath, you decrease the overall temperature of the bath solution. If you use 70% Ethanol, tissue sections will freeze evenly but more slowly than if you use 100% Ethanol. When precipitating RNA, DNA, or proteins, the Ethanol Solution can contain 70% to 100% Ethanol.
Western Blot Protocol
A widely used method for the detection of specific proteins following electrophoresis.
Principle: the protein sample is electrophoresed on a polyacrylamide gel and blotted to a membrane. The membrane is incubated with the antibody to the specific protein. Further, the secondary antibody, labeled with an enzyme such as HRP (or ALP) is added and reacted.
Chemiluminescence detection of the specific protein can be done by applying chemiluminescent substrate of the enzyme.
Protein Analysis by SDS – PAGE
Preparation of SDS-Polyacrylamide Mini Gels
The percentage of the resolving gel determines the molecular weights of proteins which can be resolved. 12% is a good generic gel to use and will resolve proteins ranging from 15kDa to 200 kDa with emphasis on resolving proteins from 15 kDa to 100 kDa.
The acrylamide content of the gel is determined by the analyzed protein size. The preparation of the separating and stacking components of the gel is based on the following chart:
| | Separeting gel | Stacking gel | |||
| % acrylamid | 10% | 12.5% | 15% | 5% | % acrylamid |
| 1.5M Tris pH 8.0 | 2.5 ml | 2.5 ml | 2.5 ml | 0.38ml | 1M Tris pH 6.8 |
| 30% acryl/0.8% bis acrylamide | 3.3 ml | 4 ml | 5 ml | 0.67ml | 30% acryl/0.8% bis acrylamide |
| SDS 20% | 0.05ml | 0.05ml | 0.05ml | 0.015 ml | SDS 20% |
| H2O | 4 ml | 3.3 ml | 2.3 ml | 2.7 ml | H2O |
| APS 10% | 0.1ml | 0.1ml | 0.1ml | 0.03ml | APS 10% |
| TEMED | 0.005ml | 0.005ml | 0.005ml | 0.003ml | TEMED |
Before pouring the separating and stacking gel seal bottom of plates with 1ml sealing gel
Sealing Gel Recipe
500ul gel solution (acrylamide:Bis-acrylamide 29:1)
5ul 10% APS
5ul TEMED (N,N,N’N’-tetramethylene-ethylenediamine)
- Pour separating gel
- Pour 1ml isopropanol on top
- Let separating gel solidify (~30min)
- Pour stacking gel and immediately insert comb
- Let stacking gel solidify (~30min)
- Gels can be used immediately or stored @ 4C wrapped with wet tissue paper and saran wrap (to avoid drying)
Preparation of Protein Samples
- Lyse cell pellet with 1ml/0.1gr (wet weight) lysis buffer. For HES cells use 1X lysis solution (50 mM Tris, pH 8.0, 150 mM NaCl, 1% (wt/vol) Triton X-100, 0.1% (wt/vol) SDS, 1% (wt/vol) deoxycholate) supplemented with a cocktail of protease inhibitors (Roche).
- Mix protein samples with 2X protein sample buffer and load onto gel
- Perform electrophoresis @ 35–45 mΑ for 1hr in protein electrophoresis buffer
Tris-Glycine (Protein Electrophoresis) Buffer
Tris base 3 g 6 g 9 g 12 g
Glycine 14.2 g 28.4 g 42.6.g 56.8 g
10% SDS 10 ml 20 ml 30 ml 40 ml
H2O to 1L to 2L to 3L to 4L
- Unless you will be proceeding to blotting, stain gel with Coomasie Brilliant Blue [1lt: 0.25 gr Coomasie Brilliant blue R250, 100ml acetic acid, 900ml H2O/MeOH 1:1] for 10 min -15 min
- Remove stain by o/n shaking in 30% MetOH , 10% acetic acid solution
- Photograph, dry and store gel
Protein Blotting
- Transfer gel to tray containing cold protein transfer buffer and incubate for 15min
- Handle PVDF membrane with forceps and not hands since this will cause high background
- Mark the membrane for orientation
- Prewet PVDF membrane with 100% MetOH and then blotting buffer and the buffer paper and sponges with blotting buffer
- Assemble electrotransfer device and place in ice bucket filled with ice
- Perform electrotransfer @ 0.4A for 2hrs
12.5X Protein Transfer Buffer (0.8L)
30 gr Tris Base
144 gr Glycine
H2O to 800 ml
1X Protein Transfer Buffer (1L)
80 ml 12.5X protein transfer buffer
720 ml dH2O
200 ml methanol
1000 ml total
Remove PVDF membrane from apparatus, place on a piece of filter paper face-up and let it air-dry o/n (membrane may be left dry for several days)
Detection
- Activate PVDF membrane by immersing in 100% MetOH for 15” and transfer to container with 50ml blocking buffer
- Incubate for 1hr @ RT
- Incubate with appropriate volume of primary antibody (diluted with hybridization/blocking buffer) for 1 hr @ RT
- Wash off primary antibody by performing 3, 15-min washes with 150ml wash buffer each time @ RT
- Incubate with appropriate volume of secondary antibody (diluted with hybridization buffer) for 1 hr @ RT
- Wash off secondary antibody by performing 3, 15-min washes with 150ml wash buffer each time @ RT
- Wash membrane once with PBS (membrane can be left in PBS until ready to proceed with detection)
- Place membrane face-up on a piece of saran-wrap on bench
- Add detection solution onto membrane
- Handling membrane with forceps touch corner on tissue to remove excess solution
- Wrap membrane in saran-wrap and place in cassette (use tape to immobilize membrane)
- Expose film to membrane using various exposure times
Reagents and Buffers
Blocking/Hybridization Buffer:
Dissolve 5gr fat-free milk in powder form in 100ml PBS (final concentration 5% milk in PBS)
Washing Buffer:
Dissolve 2.5ml concentrated NP-40 in 500ml PBS (final concentration 0.5% NP-40 in PBS)
Suggested Materials (2007)
Molecular marker (BIORAD #161-0375 Precision Plus Protein Kaleidoscope Standards, 500 µl, 50 applications, $123.00)
Protein Loading buffer (BIORAD #161-0737
Laemmli Sample Buffer, 30 ml, $13.00)
Western Apparatus
Immun-Blot PVDF Membrane (BIORAD #162-0174, $70.00)
Mini Trans-Blot Electrophoretic Transfer Cell, includes 2 gel holder cassettes, 4 fiber pads, modular electrode assembly, Bio-Ice cooling unit, lower buffer tank, lid with cables, instructions (BIORAD #170-3930, $435.00)
Fiber Pads (BIORAD #170-3933, $23.00)
1.5 mm Casting Module (BIORAD #165-3340)
15-well Combs (BIORAD #165-3366)
Tris-HCl Ready Gel Precast Gel, 12% resolving gel, 4% stacking gel, 10-well, 30 µl (BIORAD #161-1102EDU, $9.25)
Mini-PROTEAN 3 Electrophoresis Module (BIORAD #165-3302)
Blocking Buffer
Blotting-Grade Blocker Fat-free milk [BIORAD #170-6404]
Protein Electrophoresis/ Transfer Buffer
Trizma® base (SIGMA #93362, 250G, $73.90)
Glycine (SIGMA #G8898-500G, $38.00)
SDS (SIGMA #L3771-500G, 163.00)
Chemiluminescence Detection Kit
ECL Plus Western Blotting Detection Reagents (Amersham Biosciences, #RPN2132, $230.00)
Film & Cassette
Hyperfilm ECL (Amersham Biosciences, #RPN1674K)
Antibodies
Secondary:
aRabbit (GE Healthcare): dilution factor: 1/4000
aGoat (Jackson Immunoresearch) (antibody is rehydrated with 0.5ml d.H2O and additionally 0.5ml glycerol is added and Ab stored @ 4oC): dilution factor: 1/10.000
Western Blot Stripping Protocol
Stripping Buffer Recipe:
100mM 2-mercaptoethanol (280 ul 2-mercaptoethanol / 40 ml total volume)2% (w/v) SDS62.5 mM Tris, pH 6.7
Submerge membrane in stripping buffer and incubate @ 60oC for 1 hr with occasional agitation
Western Blot - Immunodetection
- Following electrotransfer of proteins from gel to blot and overnight drying of the blot, activate PVDF membrane by immersing in 100% MetOH for 15” (skip this step when re-blotting after stripping, in which case blot has been kept wet)
- transfer to appropriate container with 50ml blocking buffer*
- Incubate for 1hr @ RT while shaking mildly
- In the meantime dilute the primary antibody with blocking buffer
- Incubate blot with appropriate volume (usually no more than 10ml) of primary antibody diluted with blocking buffer for 1 hr @ RT while shaking mildly
- Wash off primary antibody by performing 3, 15-min washes with at least 150ml wash buffer** each @ RT while shaking
- Incubate with appropriate volume of secondary antibody (usually no more than 10ml) diluted with hybridization buffer for 1 hr @ RT while shaking mildly
- Wash off secondary antibody by performing 3, 15-min washes with 150ml wash buffer each time @ RT
- Wash membrane once with PBS (membrane can be left in PBS until ready to proceed with detection)
- Place membrane protein side-up on a piece of saran-wrap on bench
- Add detection solution onto membrane and incubate
- Handling membrane with forceps touch corner on tissue to remove excess solution
- Wrap membrane in another piece of saran-wrap, trim wrap and place in cassette (use tape to immobilize membrane)
- Expose film to membrane using various exposure times and develop films
Buffers
* Blocking/Hybridization Buffer:
Dissolve 5gr fat-free milk in powder form in 100ml PBS (final concentration 5% milk in PBS)
** Washing Buffer:
Dissolve 2.5ml concentrated Tergitol (NP-40) in 500ml PBS (final concentration 0.5% NP-40 in PBS)
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