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Preparation of Tissue Samples for SDS PAGE and Detection of Proteins by Immunoblot
Contributor: The Molecular Profiling Initiative of the National Cancer Institute
URL: Molecular Profiling Initiative
 
Overview
This protocol describes the preparation and analysis of proteins from microdissected cells. The cells have been isolated by laser capture microdissection (see Protocol ID#2237) and are immobilized on a thermoplastic membrane. The cells are lysed with the addition of SDS-PAGE Loading Buffer and trituration. The released proteins are fractionated by electrophoresis on a Polyacrylamide gel and transferred to a solid membrane. The membrane is probed with antibodies specific for the protein of interest and subjected to chemiluminescent detection with horseradish peroxidase (HRP)-conjugated secondary antibodies.
 
Procedure
A: Staining and Laser Capture Microdissection

1. Place a tablet of the Protease Inhibitor Cocktail (Roche) into each of the staining dishes and stain the 8 to 12 μm frozen sections of tissue as described in Protocol ID#2233 (see Hint #1).

2. Microdissect 1,000 to 2,000 cells from a stained frozen section. To minimize protein degradation, limit the time of microdissection to 30 min or less per slide (see Hint #2).

B: Protein Separation by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

1. Pipette 10 to 20 μl of SDS-PAGE Loading Buffer onto the membrane containing the microdissected cells that have been isolated as described in Protocol ID#2237. Continue to triturate buffer on top of the cells for 2 to 3 min in order to dissolve the cells in the buffer.

2. Transfer the lysed cells in loading buffer to a screw-capped tube and boil the sample for 3 min.

3. Centrifuge the tube briefly to collect any liquid that may have condensed at the top of the tube.

4. Prepare an SDS-Polyacrylamide gel as described in Protocol ID#455.

5. Load the entire sample containing the proteins extracted from the isolated cells onto the SDS-Polyacrylamide gel. Load a Molecular Weight Protein Standard (e.g., Multimark, Novex) in one lane.

6. Electrophorese the proteins at 110 Volts for 1.5 to 2 hrs as described (see Protocol ID#455).

C: Electrophoretic Transfer of Proteins to a Membrane (Nylon, PVDF or Nitrocellulose)

1. After electrophoresis, wash the acrylamide gel in 1X Transfer Buffer (Novex) for 30 min (see Hint #3).

2. Prepare the membrane and support media as follows:
   Soak four to five sponges in Transfer Buffer
   Soak two pieces of Whatman 3MM filter paper in Transfer Buffer
   Soak the membrane briefly in Transfer Buffer
   (for Polyvinyldifluoride [PVDF] membranes, soak the membrane briefly in 100% (v/v) Methanol followed by 1X Transfer Buffer)

3. Assemble the "sandwich" on top of the open transfer cage in the following sequence:
   Two sponges
    One filter paper
   The Polyacrylamide gel
   The Membrane
   One filter paper
   Two to three sponges

4. Close the transfer cage to complete the "sandwich" and immerse the assembly into the transfer chamber. Ensure that the cage is oriented such that the side of the gel contacting the membrane is facing the positive electrode (see Protocol ID#70).

5. Fill the chamber with Transfer Buffer and electrophorese the proteins from the gel to the membrane at 40 Volts for 2 hr.

D: Primary and Secondary Antibody Incubations

1. Remove the membrane after transfer and place it in 50 to 100 ml of Blocking Solution for 30 min at room temperature.

2. Wash the membrane three times for 10 min in 50 to 100 ml of Wash Solution I.

3. Incubate the membrane with appropriately diluted primary antibody overnight in enough Wash Solution I to cover the membrane and allow gentle agitation at 4°C (see Hint #4).

4. Wash the membrane three times for 10 min in 50 to 100 ml of Wash Solution I.

5. Incubate with an appropriately diluted horseradish peroxidase-conjugated secondary antibody for 1 hr with gentle agitation at 4°C (see Hint #5).

6. Wash the membrane three times for 10 min in 50 to 100 ml of Wash Solution II.

E: Detection

1. For the chemiluminescent reaction, incubate the membrane in a mixture of an equal volume of Luminol and an equal volume of oxidizing reagent for 5 min at room temperature (see Protocol ID#1021 and Protocol ID#70).

2. Wrap the membrane in plastic wrap, attach phosphorescent markers to the edges of the membrane on top of the plastic wrap, and perform detection with autoradiography (see Protocol ID#9036).

Solutions
PBS (1X)   1.44 g Sodium Phosphate, Dibasic (Na2HPO4)
0.24 g Potassium Phosphate, Monobasic (KH2PO4)
Adjust pH to 7.4 with HCl
0.2 g KCl
Add H2O to 1 liter
8 g NaCl
Store at room temperature
800 ml of ddH2O
Autoclave to sterilize
Wash Solution II (for 1 Liter)   900 ml of ddH2O
100 ml of 10X PBS
1 ml of Tween-20
Wash Solution I (for 1 Liter)   1 g Ovalbumin (Sigma)
1 g Non-Fat Dry Milk
900 ml of ddH2O
100 ml of 10X PBS
1 ml of Tween-20
Blocking Solution   Blocking solution containing casein (Pierce)
Transfer Buffer   Tris-Glycine Transfer Buffer (25X) (Novex)
Molecular Weight Protein Standard   e.g., Multimark, Novex
SDS-PAGE Loading Buffer (1X)   Add DTT right before use
2% (w/v) SDS
10% (v/v) Glycerol
0.1% (w/v) Bromophenol Blue
Can be stored at room temperature without the DTT
100 mM DTT
50 mM Tris-Cl, pH 6.8
Protease Inhibitor Cocktail Tablets   Roche
 
BioReagents and Chemicals
Sodium Chloride
Methanol
Milk, Non-Fat Dry
Ovalbumin
Potassium Phosphate, Monobasic
Hydrochloric Acid
Tris-Cl
Luminol
Secondary Antibody, Horseradish peroxidase-conjugated
DTT
Glycerol
Tween-20
Bromophenol Blue
SDS
Primary Antibody
Sodium Phosphate, Dibasic
Potassium Chloride
 
Protocol Hints
1. Staining with Hematoxylin decreases subsequent protein recovery and should be avoided if possible. If Hematoxylin is required for visualization during microdissection, then isolate approximately twice as many cells to ensure adequate recovery.

2. For low abundance proteins, more cells may be required for detection.

3. For some low molecular weight proteins, omit this step as the proteins may diffuse out of the gel during this wash.

4. The optimal dilution of primary antibody requires empirical determination. Cover the container with plastic wrap to minimize evaporation.

5. The contributor of this protocol recommends a 1:20,000 dilution of the secondary antibody as a starting point for optimizing the dilution. The optimal dilution requires empirical determination. Alternatively, biotinylation of the secondary antibody increases sensitivity through detection with a streptavidin-peroxidase conjugate.

 
Citation and/or Web Resources
1. Molecular Profiling Initiative.

   


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