of Multi-Protein Complexes (MPCs)
· What are MPCs: MPCs are a group of two or more associated
polypeptide chains. Two kinds of MPCs can be distinguished: Constitutive,
abundant MPCs such as receptors or transcription factors; and signal-induced,
transient, low copy number MPCs. These complexes typically play a crucial role
in signaling. Identification and analysis of MPCs requires their separation
under native conditions.
· BN-PAGE: Blue native polyacrylamide gel electrophoresis (BN-PAGE)
can be used for one-step isolation of multi-protein complexes (MPCs) from
biological membranes/organelles and total cell and tissue homogenates under
native conditions. BN-PAGE is often used for the study of MPCs as it can
provide information about the size, number, protein composition, stoichiometry,
or relative abundance of MPCs.
o It can be used to
determine native protein masses, oligomeric states, and identify physiological
o It has a higher resolution for separation than
gel filtration or sucrose density ultracentrifugation.
o It can be used to analyze abundant, stable
MPCs from 10 kD to 10 MD.
o It allows the determination of the size, the
relative abundance, and the subunit composition of an MPC, in contrast to,
immunoprecipitation and two-hybrid approaches.
o It is useful for determining how many
different complexes exist that share a common subunit, whether free monomeric
forms of individual subunits exist, and whether these parameters change upon
Provided below is a detailed protocol for the
separation of MPCs from different samples and their analysis. This protocol can
also be used for separation of pre-purified MPCs by one-dimensional BN-PAGE or
by two-dimensional BN-PAGE followed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
1. Sample preparation for BN-PAGE:
Note about detergents: Before preparing the samples for BN-PAGE, the optimal detergent and
its appropriate concentration that will preserve the MPCs yet solubilize the
cells should be determined. Non-ionic detergents are considered best for
maintaining MPC stability. Commonly used detergents that can be tested include
digitonin (0.5 to 1%), Triton X-100 (0.1 to 0.5%), Brij 96 (0.1 to 0.5%), or
dodecylmaltoside (0.1 to 0.5%). Other
detergents can also be used [information about detergents can be found online
or on request]. Even if soluble MPCs are being analyzed, detergents must be present in the dialysis step to prevent aggregation during the stacking step of the gel run.
Samples for BN-PAGE may be prepared from several sources
such as total cell lysates, cellular organelles and membranes. A basic protocol
for MPC sample preparation from total cell lysates or from lysates of tissue
samples for BN-PAGE analysis is provided below. The details of cell culture
will be specific to the cells used for the experiment and are therefore not
1a. Preparation of total cell lysates
1. Harvest 2 x 106 cells and pellet by centrifugation at 350g for
5 min at 4°C.
Note: Suspension cells can be harvested by
centrifugation; cells that grow attached to the culture dishes should be
released from the dishes with 0.5 mM EDTA (avoid trypsin, because it digests
extracellular proteins). Tissues should be homogenized with a Dounce
homogenizer in PBS (Mix 1).
2. Wash the cell pellet three times with 0.5 ml of
ice-cold PBS (Mix 1), and pellet after each wash by centrifugation at 350g for 5 min at 4°C.
3. Resuspend the cell pellet at 2 x
106 cells per 100 µl of ice-cold BN-Lysis Buffer
4. Incubate on ice for 15 min.
5. Centrifuge at 13,000g for 15 min
6. Melt a hole in the cap of a microcentrifuge
tube using a hot Pasteur pipette, and then place the tube on ice to chill.
7. Transfer the supernatant from step 5 into the
chilled tube with the hole in the cap.
8. Place a dialysis membrane with forceps over the opened tube and close the cap
(F2, B and C).
Note: Ensure that there are no folds or
tears in the dialysis membrane.
9. Seal the cap carefully with Parafilm.
10. Invert the tube and centrifuge upside-down at
the lowest speed possible
in the adaptor cavity for 50-ml conical tubes in a cell culture centrifuge for
10 s at 4°C.
Note: Remove the inverted tube from the centrifuge using large tweezers to avoid turning the tube right side up.
11. Prepare a 100-ml beaker with cold BN-Dialysis
Buffer (Mix 4) and a magnetic stirrer. Use at least 10 ml of BN-Dialysis Buffer
per 100-µl sample.
12. Affix the tube with tape upside-down inside
the beaker and remove air bubbles from the hole beneath the cap using a bent Pasteur pipette.
13. Switch on the magnet stirrer and leave it for 6 hours or overnight in
the cold room. Check occasionally to ensure
that stirring is not creating air bubbles at the dialysis membrane.
14. Collect the dialyzed cell lysate in a new
chilled microcentrifuge tube.
It might sometimes be necessary to increase the likelihood of
detection of less abundant MPCs. For this purpose, we provide two alternative sample preparation
methods that allow enrichment
of samples for MPCs with i. phosphotyrosine-containing proteins and ii. MPCs
associated with cell membranes or organelles.
1b. Enrichment of MPCs containing phosphotyrosine residues
Immunoprecipitation methods commonly used for enrichment of
proteins cannot be combined with BN-PAGE, because
elution of MPCs under native conditions from the antibody-coupled beads is
impossible. MPCs with components that are phosphorylated on tyrosine residues may be immunopurified using
antibodies against phosphotyrosine. The MPCs are then eluted with an excess of
phenylphosphate, which competes with phosphotyrosine for binding to the antibody. Provided below is a
detailed protocol for preparation of phosphotyrosine-enriched samples.
Alternatively, affinity-purification protocols, such as the tandem affinity
purification (TAP-tag) method,
that allow native elution
of the proteins from the affinity matrix could also be used.
1. Harvest 2 x 107 cells and pellet by centrifugation at 350g for
5 min at 4°C.
Note: Cells may be stimulated with
ligands, agonists, antagonists, or other conditions, depending on the
2. Wash the cell pellet once with 1 ml of ice-cold PBS (Mix 1) and pellet by centrifugation at 350g for 5 min at 4°C.
3. Resuspend the cell pellet at 2 x
107 cells per 1 µl of ice-cold Lysis Buffer
4. Incubate for 15 min on ice.
5. Centrifuge at 13,000g for 15 min
6. Transfer the supernatant from step 5 into a
new chilled tube.
7. Add 10 µl of beads coupled to antibodies
against phosphotyrosine residues and incubate on a rotating wheel for 2 hours
to overnight in the cold room.
8. Centrifuge at 400g for 2 min at 4°C.
9. Wash the beads three times with 1 ml of ice-cold BN-Dialysis Buffer (Mix 4), centrifuging 400g for 2 min at 4°C
10. Add 40 µl of ice-cold BN-Dialysis Buffer
Containing Phenylphosphate (Mix 6) and resuspend the beads using a 20-µl
pipette tip that has been cut at the narrow end to make the opening larger.
Resuspend beads every 5 min for 30 min while incubating on ice.
Note: If desired, proteins may be
dephosphorylated by adding one unit of alkaline phosphatase to the eluate
during the last 5 min of the elution procedure.
Centrifuge at 400g for 2 min at 4°C and collect the
supernatant (eluate) in a new, chilled tube.
Note: These samples do
not have to be dialyzed and are ready for BN-PAGE.
1c. Preparation of membranes for BN-PAGE
MPCs that are associated with
cell membranes or inside organelles can be enriched before preparing the lysate
for analysis by BN-PAGE.
1. Prepare membrane fractions of cells using standard protocols.
2. Wash the membrane pellet once with 0.5 ml of ice-cold BN-Lysis Buffer (Mix 3) without detergent.
3. Resuspend the membrane pellet completely without generating air
bubbles in ice-cold BN-Lysis Buffer
(Mix 3), including detergent. Use the equivalent of 4 x 107 cells per 100
ul of BN-Lysis Buffer.
4. Incubate for 1 hour at 4°C, resuspending the pellet every 15 min.
5. Centrifuge at 20,000g for 10 min at 4°C.
6. Collect the supernatant (membrane lysate) in a new, chilled microcentrifuge tube.
Note: These samples do not have to be
dialyzed and are ready for BN-PAGE.
2. BN-Gel preparation
Carry out gradient gel pouring at room temperature with a gradient
mixer. Because of its high glycerol content, the gel mix with the
higher-percentage (15%) acrylamide-bisacrylamide is heavier than the
low-percentage (4%) gel. This density difference aids in establishment of a
uniform gradient between the glass plates. Gloves must be worn because polyacrylamide is highly
Note: Precast BN-gels and buffers are commercially available from Invitrogen (NativePAGE Novex Bis-Tris Gel System).
1. Place the gradient maker on a
stir plate and attach to a peristaltic pump. Close the channel using the valve
and close the tubing with a clamp.
2. Attach a syringe needle to the
end of a piece of flexible tubing that comes out of the peristaltic pump and
place the needle into the top, between the two glass plates of the gel apparatus. Place the needle
close to the bottom and raise it slowly as
the gel pours.
3. Prepare 4% and 15% Separating
Gels (Mixs 9 and 10), adding APS and TEMED only immediately before use.
Note: The volumes of the two gel solutions
combined should be exactly equal to the volume required to fill the space
between the glass plates to the required height.
4. Pour these gel solutions into the
corresponding cylinders of the gradient mixer (4% into the “low” and 15% into the “high”
5. Open the valve and force out
the air bubble inside the channel
connecting the two gel reservoirs by pressing over the left cylinder with your thumb.
Switch on the
pump to 5 ml per minute, remove the clamp, and allow the
gel to slowly flow between
the glass plates. Ensure that the needle is always above the height of the liquid so that the gradient is not disturbed
7. Allow all liquid to enter the gel
apparatus, and then overlay gently
with isopropanol. Allow the
gel to polymerize for at least 30 min at room temperature.
8. Clean the pouring apparatus with dH2O (do not
9. Remove the isopropanol, wash with dH2O, and remove the dH2O carefully with a slip of absorbent paper without touching the gel.
10. Prepare a 3.2% Stacking Gel (Mix 11), adding APS and TEMED only immediately before use.
11. Pour the stacking gel on top of the separating gel
and immediately introduce the comb between the glass plates, avoiding bubbles at the interface
between the gel solution and the comb.
Allow the gel to polymerize.
Note: Make sure that at least 0.5 cm (for
5-ml minigels) or 2.5 cm (for large, 30- to 50-ml gels) of stacking gel remain
between the comb and the separating gel.
12. Remove the comb slowly, pulling it out at an angle to
the plane of the gel. This allows air
to enter the pockets rapidly, which improves the
quality of the wells.
3. Separation of the
prepared sample of MPCs by BN-PAGE
Coomassie blue is present in the solution at the cathode that is
overlaid onto the samples that have been added to the wells. This dye interacts
with the MPCs inside the wells of the gel and enters the gel during
electrophoresis, preventing aggregation of proteins in the stacking gel. Once
the samples are prepared, the remainder of this part of the procedure should be
performed at 4°C. It is recommended to boil an aliquot of the sample with SDS
to disrupt the MPCs as a control and loading this control also on the BN-PAGE.
1. Boil an aliquot of the sample, to be used as a control, in 1% SDS
for 5 min to dissociate all MPCs. Leave one
lane empty between this control and the “non-SDS samples.”
2.. Load 1 to 20 µl of sample in the dry wells,
before adding the Cathode Buffer (Mix 12).
Note: If the sample contains
phosphorylated proteins and the phosphorylation is to be preserved, add 1/100th
volume of 100x Pervanadate (Mix
13) to the dialyzed lysate.
3. Load 10 µl of Marker Mix (Mix 14).
Note: Only ferritin is visible during the
electrophoresis due to its brown color. The other markers will be visible
following Coomassie or silver staining.
4. Overlay the samples in each well with Cathode Buffer (Mix 12).
5. Fill the inner chamber with Cathode Buffer (Mix 12) and the outer and lower chambers with Anode Buffer (Mix 15).
6. Apply voltage to a minigel at 100 V or a large gel at 150 V, until the samples have entered the separating gel.
7. Increase the voltage to 180 V (minigel) or 400 V (large gel) and run until the dye front reaches the end of the gel.
Note: The gel run takes between 3 and 4
hours for a mini-gel, and between 18 and 24 hours for a large gel.
4. Second Dimension
After performing the
first-dimension BN-PAGE, it is possible to run a second-dimension SDS-PAGE to
separate each MPC into its components. The protocol for this is provided below.
1. Prepare a standard SDS-PAGE gel with a single large lane and one lane for
molecular weight markers.
Note: To make it easier to load the BN-PAGE gel slice onto the second-dimension gel, tape (Tesafilm or Cellotape) the spacers and comb of
the SDS-PAGE gel. This
results in a slightly thicker gel, which is sufficient to allow
the BN-PAGE gel slice to slip between the plates and into the well easily, but still remain fixed between the glass plates.
2. Remove the BN-PAGE gel
in the plates from the electrophoresis apparatus and gently pry up one plate.
3. Cut out the lane of the BN-PAGE gel containing the proteins
of interest and remove the
4. Place the BN-PAGE gel
slice in SDS Sample Buffer (Mix 16) (5 ml for a minigel slice) in a small dish
or cell culture plate, and incubate for 10
5. Boil the BN-PAGE gel slice briefly (not more than 20 s) in the microwave.
Note: Excessive boiling will cause the gel
slice to shrink.
6. Continue incubating the BN-PAGE gel slice in the hot SDS Sample Buffer (Mix 16) for another 15 to 20 min.
7. Load the BN-PAGE gel
slice over the stacking gel
of the SDS-PAGE gel
and overlay the slice with
SDS Sample Buffer (Mix 16).
8. Perform electrophoresis according to standard protocols.
5. Visualization of MPCs
Several methods are available for
visualizing the protein complexes. The protein constituents of MPCs that have been isolated by BN-PAGE or
second dimension SDS-PAGE can
be analyzed by Coomassie
blue staining or silver staining. Coomassie blue is a good choice for highly
abundant MPCs (mg amounts), whereas silver staining is good for visualization
of 50 to 1000 ng amounts of the protein of interest. These methods are standard, and the details are not
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