| Contributor: |
The Laboratory of Paul Kaufman at the University of California, Berkeley and
the Lawrence Berkeley National Laboratory
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| This method allows for separation of differently acetylated histones and some histone subtypes. There are a number of isoforms (subtypes) of the histone proteins H2A, H2B, H3, and H4. In addition, these proteins can be modified by acetylation and phosphorylation. Triton-Acetic Acid-Urea (TAU) gels can separate the modified variants and the subtypes of histone H2A, H2B, and H3 based on charge and mass. |
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A. Casting TAU Gels
1. Set up gel plates using spacers no thicker than 0.75 mm. The contributor finds that samples run better on larger format gels (approximately 20 cm x 30 cm).
2. For 1 large 15% Acrylamide Gel, combine (see Hint #1):
7.5 ml of 40% (w/v) Acrylamide (see Hint #2)
0.9 ml of 2% (w/v) Bisacrylamide (see Hint #2)
7.28 g of Urea
0.74 ml of 10% Triton X-100
1.0 ml of Glacial Acetic Acid
Add ddH2O to a final volume of 20 ml
3. After the Urea dissolves, filter the solution through a 0.45 um filter.
4. Degas the solution under house vacuum.
5. Add 128 μl of 10% Ammonium Persulfate and 100 μl of TEMED to initiate polymerization of the acrylamide.
6. Immediately pour the Acrylamide solution into the plates, leaving room for the stacking gel, and overlay with water-saturated Butanol. The gel will take up to 2 hours to polymerize.
7. Prepare a 6% Acrylamide Stacking Gel mix by combining:
0.6 ml of 40% (w/v) Acrylamide
80 μl of 2% (w/v) Bisacrylamide
1.46 g of Urea
75 μl of 10% Triton X-100
0.2 ml of Glacial Acetic Acid
Add ddH2O to a final volume of 4 ml
8. Pour off the Butanol from the top of the polymerized running gel. Rinse the top of the gel with a small amount of stacking gel mix.
9. Add 26 μl of 10% Ammonium Persulfate and 20 μl of TEMED to start polymerization. Pour the Acrylamide Stacking Gel to within a few millimeters of the top of the gel. Don't use a comb (see Hint #3). Overlay with water-saturated Butanol.
10. After polymerization of the stack, remove the Butanol and rinse the top of the gel with 5% Acetic Acid.
11. Place the gel in a running tank and use 5% Acetic Acid as the running buffer.
B. Preparation of Gel by Preliminary Electrophoresis
1. Overlay the gel with approximately 1 ml of Overlay Solution.
2. Electrophorese the gel overnight at 100V (in the cold room to help dissipate the heat). The current should decrease from 20 mA initially to 2 mA. Be sure to reverse the leads as compared to regular SDS-PAGE. The red lead should be connected to the top of the gel, the black lead to the bottom. In the presence of Acetic Acid, all proteins become negatively charged, and migrate toward the negative charge of the black lead. When the samples are added later, proteins with more positive charges (e.g. unacetylated Histone H4) will migrate more quickly than those with less positive charge (e.g. acetylated Histone H4, in which basic lysine amino groups are neutralized by acetylation).
3. Clean off the top gel surface using a syringe filled with fresh 5% Acetic Acid.
4. Load 0.5 ml of Scavenging Solution.
5. Electrophorese the gel at 200 V for 2 hr. The tracking dye won't run into the gel this time. This step removes the free radicals that can modify proteins and alter their migration.
6. Clean off the top gel surface using a syringe filled with fresh 5% Acetic Acid.
7. Load a second 0.5 ml of Scavenging Solution.
8. Electrophorese the gel at 200 V for 1 hr.
9. Clean off the top gel surface using a syringe filled with fresh 5% Acetic Acid.
10. Load 0.5 ml of Protamine Solution.
11. Electrophorese the gel at 200 V for 1 hr. These highly basic proteins block non-specific interactions between the gel matrix and the histones. The tracking dye will now migrate.
C. Electrophoresing Protein Samples on the TAU Gel
1. Dissolve TCA-precipitated material directly into Protamine Solution, or dissolve the concentrated stock in several volumes of the same solution. No boiling of the samples is necessary (or desirable; it causes carbamylation by the urea).
2. Clean off the top gel surface using a syringe filled with fresh 5% Acetic Acid.
3. Insert a comb into the stacking gel until the bottom of the teeth are clearly immersed in the polyacrylamide, with no space underneath. A little upward bulging of the polyacrylamide between the teeth is fine.
4. Load the protein samples with a Hamilton syringe into the spaces between the teeth using sample volumes of less than 8 ul. Be careful not to load too much protein or smeary bands will result (see Hint #4).
5. Load 1 μl of Cytochrome C as a visible marker. It will run approximately with histone H4 with this gel recipe as a faint brown band. Leave several lanes empty between the Cytochrome C marker and any important samples.
6. Electrophorese the gel at 200 to 250 V for approximately 3000 V.hr, until the Cytochrome C is approximately three-quarters down the gel.
7. The gel may be stained with Coomassie stain, like typical SDS polyacrylamide gels (see Protocol on Coomassie Staining of Protein Gels).
D. Sliver Staining of TAU Gels (Optional) (see Hint #5)
1. Stain the gel first with Cobalt-Amido Black stain by incubating gel at room temperature for more than 40 min. Gel will turn opaque black.
2. Remove the dye.
3. Destain the gel by adding 0.5 M Sulfuric Acid.
4. The gel will become lighter and the heavily loaded Cytochrome C bands will be visible within 10 min when the gel is placed on a light box.
5. Progressively destain the gel by removing solution and replacing it with 250 mM Sulfuric Acid.
6. Remove the solution and replace with 100 mM Sulfuric Acid.
7. Remove the solution and replace with 50 mM Sulfuric Acid.
8. Remove the solution and replace with ddH2O (see Hint #6).
E. Western Blotting of a TAU Gel
1. Wash the gel after electrophoresis twice for 30 min each in Wash Buffer I.
2. Wash the gel once for 30 min in Wash Buffer II.
3. Cut a piece of PVDF (Immobilon-P, Millipore) membrane to the size of the gel.
4. Pre-wet the PVDF membrane in 100% Methanol and then equilibrate in Transfer Buffer.
5. Set up a Western transfer apparatus according to the manufacturer's instructions.
6. Transfer the protein from the gel to the PVDF membrane in Transfer Buffer at 70 V for 2 hours.
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| Scavenging Solution |
| 0.02% (w/v) Pyronine Y
2.5 M Cysteamine-HCl (add from 5 M stock in water stored at -80°C; Sigma)
6 M Urea
5% (v/v) Acetic Acid
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| Overlay Solution |
| 0.02% (w/v) Pyronine Y (see Hint #7)
6 M Urea
5% (v/v) Acetic Acid
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| 5% Acetic Acid |
| Dilute from Glacial Acetic Acid (99%)
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| Transfer Buffer |
| 20% (v/v) Methanol
25 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS)-NaOH, pH 10.0
Buffer cannot be reused
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| 10% Triton X-100 |
| 10% (v/v) Triton X-100
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| Wash Buffer II |
| 5% 2-Mercaptoethanol
2% (w/v) SDS
62.5 mM Tris-HCl, pH 6.8
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| 10% Ammonium Persulfate |
| 10% (w/v) Ammonium Persulfate
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| Wash Buffer I |
| 0.5% (w/v) SDS
50 mM Acetic Acid
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| 50 mM Sulfuric Acid |
| Dilute 100 mM Sulfuric Acid 1:1 with ddH2O
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| 100 mM Sulfuric Acid |
| Dilute 0.5 M Sulfuric Acid 1:5 with ddH2O
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| 250 mM Sulfuric Acid |
| Dilute 0.5 M Sulfuric Acid 1:1 with ddH2O
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| 0.5 M Sulfuric Acid |
| Make from Concentrated Sulfuric Acid stock (18 M)
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| 1 M Cobalt Nitrate |
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| Amido Black Stain |
| Prepare in 1% Acetic Acid
2 mM Amido Black 10B, Electrophoresis Grade (also known as Napthol Blue Black; Sigma)
Immediately before use, add 0.6 ml of 1 M Cobalt Nitrate per 200 ml stain solution
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| Protamine Solution |
| 25 mg/ml Protamine Sulfate, Herring Type III (add from 50 mg/ml stock in water stored at -80°C; Sigma)
0.02% (w/v) Pyronine Y
6 M Urea
5% (v/v) Acetic Acid
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| Cytochrome C |
| 50 μg/μl Cytochrome C
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Bisacrylamide
Acetic Acid
Urea
Tris
TEMED
Ammonium Persulfate
Acrylamide
SDS
Cysteamine-HCl
Cobalt Nitrate
Butanol, Water-Saturated
Sulfuric Acid
Amido Black 10B
Methanol
3-[cyclohexylamino]-1-Propanesulfonic Acid
Triton X-100
2-Mercaptoethanol
Sodium Hydroxide
Pprotamine Sulfate, Herring Type III
Pyronine Y
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1. The contributor of this protocol has used these conditions to separate histones isolated from human cells.
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
3. No comb is used to make wells. The stack of these gels is very gooey and wells don't stand up well during all the preliminary electrophoresis. After the preliminary electrophoresis runs, wells will be made by inserting a comb backwards into the gel until the bottom of the teeth of the comb hits the top of the stack, creating "wells" between neighboring teeth. Make sure to pour the stack close enough to the top of the plates (allowing for incomplete polymerization at the top) so that the comb can touch the stacking layer. However, you will need to load the gels while the comb is still inserted into the gel, so the comb should not be inserted all of the way.
4. The contributor of this protocol suggests that more than about 2 μg of protein is too much.
5. The contributor has found that, contrary to Coomassie staining, these gels are very suitable to the conditions for silver staining. Generally silver staining is much more sensitive than Coomassie Blue staining. The staining procedure given in this section is the only one that the contributor has found to work. If you plan to not follow this particular silver stain, be sure to use a silver stain protocol that includes copper chloride and potassium permanganate. Finally, do not simply carry out long acid washes of the gel without the Amido Black present before the silver stain. There will usually be a brown background to the silver stained gels
6. Both signal and background will disappear over the course of these washes.
7. This is used as a tracking dye.
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1. Lennox RW and Cohen LH. Analysis of histone subtypes and their modified forms by polyacrylamide gel electrophoresis. Methods in Enzymology 1989; 170:532-549.
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