ELECTROPHOREESIS
CONTENT:
Principle, SDS PAGE, use and preparation of buffer, data analysis, gel preparation (key chemicals used), schematic representation.
Electrophoresis
is the migration of charged molecules in response to an electric field. Their
rate of migration depends on the strength of the field; on the net charge, size
and shape of the molecules, and also on the ionic strength, viscosity, and
temperature of the medium in which the molecules are moving. As an analytical
tool, electrophoresis is simple, rapid, and highly sensitive. It is used
analytically to study the properties of a single charged species, and as a
separation technique.
There are a
variety of electrophoretic techniques, which yield different information and
have different uses. Generally, the samples are run in a support matrix, the
most commonly used being agarose and polyacrylamide. These are porous gels, and
under appropriate conditions, they provide a means of separating molecules by
size. We will focus on those methods used for proteins. These can be denaturing
or non-denaturing. Nondenaturing methods allow recovery of active proteins and
can be used to analyze enzyme activity or any other analysis that requires a
native protein structure. Two commonly used techniques in biochemistry are
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
isoelectric focusing (IEF). SDS-PAGE separates proteins according to molecular
weight and IEF separates according to isoelectric point. This laboratory
exercise will introduce you to SDS-PAGE.
SDS-PAGE
1. Make sure
gel plates are clean and dry. Do not get your fingerprints on them or the
acrylamide will not polymerize properly.
2. Prepare gel solutions (separating and
stacking), but do not add polymerizing agents, APS and TEMED (this would start
the polymerization).
3. Lay the comb on the unnotched plate and
mark (on the outside, using a Sharpie) about 1 cm below the bottom of the
teeth. This will be the level of the separating gel. If available, use an
alumina (opaque, white) plate, for the notched plate, as this conducts heat
away from the gel more efficiently than glass. Set up the gel plates, spacers,
and plastic pouch in the gel casting as described in the manufacturer’s
directions. When everything is completely ready, add TEMED to the separating
gel solution, mix well, and pour it between the plates, up to the mark. Wear
gloves if you pour directly from the beaker. You can also use a disposable
pipette. Work quickly or the solution will polymerize too soon. Carefully layer
isopropanol (or water-saturated butanol) on top of acrylamide so it will
polymerize with a flat top surface (i.e., no meniscus). Do this at the side and
avoid large drops, so as not to disturb the gel surface. When the leftover
acrylamide in the beaker is polymerized, the acrylamide between the plates will
also be ready.
4. If you
are running the gel on the same day, prepare samples while the acrylamide is
polymerizing. Otherwise, wait until you are ready to run the gel.
(i) You will
need a sample of each unknown substance, plus the molecular weight standards.
Prepare samples in screw-cap microcentrifuge tubes. The protein content should
be at 1–50 mg in 20–30 mL sample. The total sample volume that can be loaded
depends on the thickness of the gel and the diameter of the comb teeth. For
Genei apparatuses, this is ~ 30 mL/well. To prepare the sample, mix 7–10 mL of
the sample (depending on protein concentration) +20 mL 2X sample buffer
containing 10% b-mercaptoethanol (BME). Use the BME in the hood - it stinks!
For dilute samples, mix 40 mL of the sample and 10 mL 5X sample buffer and add
2 mL of BME. Heat to 90°C for 3 minutes to completely denature proteins. It is
important to heat samples immediately after the addition of the sample buffer.
Partially denatured proteins are much more susceptible to proteolysis and
proteases are not the first proteins to get denatured. (Heat samples to 37°C to
redissolve SDS before running the gel if samples have been stored after
preparation).
(ii) If you
want the proteins in the sample to retain disulfide bonds, do not add BME. If
both reduced and nonreduced samples will be run on the same gel, leave at least
3–4 empty wells between samples, since the BME will diffuse between wells and
reduce proteins in adjacent samples.
(iii) MW
Stds: 7 mL of Rainbow stds +10 mL of sample buffer (do not make in advance).
Heat to 37°C before use.
5. After the separating gel has polymerized,
drain off the isopropanol. Add TEMED to the stacking gel solution, pour the
solution between the plates, and insert the comb to make wells for loading
samples. The person putting in the comb should wear gloves. Keep an eye on this
while it’s polymerizing and add more gel solution if the level falls (as it
usually does), or the wells will be too small.
6. after polymerization, do not cut the bag;
we reuse them. The gel may be stored at this point by taping the bag shut to
prevent drying. When ready to run the gel: mark the position of each well,
since they are difficult to see when full.
7. Remove
comb and rinse wells with running buffer. See the manual directions for setting
up the gels in the buffer chambers. The apparatus can run 2 gels simultaneously.
There is a blank plate to use when running only one. Fill the upper chamber
with running buffer first and check for leaks. Adjust the plates if necessary.
Load the samples using a micropipettor with gel-loading tips (these are longer
and thinner than the normal tips). This will be demonstrated. Do not load
samples in the end wells. Make sure to write down which sample was loaded in
each well.
8.
Electrophoresis (takes 1–2 hours). Connect the gel apparatus to the power
supply and run at 15 mA/gel until the tracking dye (blue) moves past the end of
the stacking gel. Increase the current to 20–25 mA/gel but make sure the
voltage does not get above 210 V. Run until the blue tracking dye moves to the
bottom of the separating gel. For the BioRad apparatus do not exceed 30 mA,
regardless of the number of gels. 9. Disassemble the apparatus and carefully
separate the gel plates using a flat spatula. Cut off the stacking gel and any
gel below the blue tracking dye. Note the color of each of the molecular weight
standards, as they will all be blue after staining. Wash 3X with distilled
water. Place the gel in a plastic staining container and add Coomassie Blue
staining solution. Keep it in this 1 hour overnight. Wash again with water. You
can wrap the gel in plastic wrap and Xerox or scan it to have a copy. The gel
may also be dried.
Data Analysis
Measure the length of the gel (since you cut off the bottom, this is the distance traveled by the dye). Measure the distance traveled by each of the molecular weight standards. Measure the distances of each unknown band. For samples lanes with many bands (serum in this exercise), measure all bands in those with just a few and the major bands in those that have many. Prepare a standard curve by plotting log MW versus relative mobility (Rf, distance traveled by protein divided by distance traveled by dye). Use this and the mobility of bands from your fractions to determine the MW of the unknown proteins. (Review standard curves from the protein quantitation lab if necessary.) MW of proteins that do not run very far into the gel or run near the dye front will not be accurate. If you have reduced and unreduced samples, compare the number of bands and MW of each to determine the number of subunits.
Gel Solutions
1. Separating gel: (15 mL, enough for
two gels) 10% acrylamide. 40% Acrylamide/bisacrylamide mix 3.55 mL. 1.5 M tris
pH 8.8, 3.75 mL, H2O 7.4 mL, 10% SDS 150 mL, 10% ammonium persulfate (APS) 150
mL (prepared fresh), TEMED 6 mL.
2. 2. Stacking gel: (5 mL) 5%
acrylamide. Compresses the protein sample into a narrow band for better
resolution. 40% Acrylamide/bisacrylamide mix 0.625 mL. 0.5 M tris pH 6.8, 1.25
mL, H2O 3.0 mL, 10% SDS 50 mL, 10% APS 50 mL, TEMED 5 mL.
3. 3. 2X sample buffer (10 mL)—store in
the freezer for an extended time.
4. 4. SDS must be at room temperature to
dissolve.
5. 5. H2O 1.5 mL, 0.5 M Tris pH 6.8, 2.5
mL, 10% SDS (optional) 4.0 mL, glycerol 2.0 mL, BPB 0.01%, b-mercaptoethanol
(optional) 0.1 mL.
6. 6. Running buffer (5L) 30 g Tris
Base, 144 g glycine, dissolve in sufficient H2O to make 1.5 L and put into
final container. Add 1.5 g SDS (Caution: do not inhale dust). When adding SDS,
avoid making too much foam, which makes measuring and pouring difficult. Final
pH should be around 8.3, but do not adjust it or the ionic strength will be too
high and the gel will not run properly. If the pH is way off, it was made
incorrectly or is old and has some contamination. The running buffer can also
be made more concentrated (5X or 10X) and diluted as needed to save bottle
space.
Electrophoresis
is defined as the separation (migration) of charged particles through a
solution or gel, under the influence of an electrical field. The rate of
movement of particle depends on the following factors.
1. The
charge of the particle.
2. Applied
electric field.
3.
Temperature.
4. Nature of
the suspended medium.
What is Gel Electrophoresis?
Gel electrophoresis is a method that separates macromolecules—either nucleic acids or proteins—on the basis of size, electric charge, and other physical properties. A gel is a colloid in a solid form. The term electrophoresis describes the migration of charged particles under the influence of an electric field. “Electro” refers to the energy of electricity. “Phoresis,” from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium. Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionizable groups and, therefore, at any given pH, exist in solution as electrically charged species, either as cations (+) or anions (–). Depending on the nature of the net charge, the charged particles will migrate to either the cathode or the anode.
How does this Technique Work?
Gel electrophoresis is a technique used for the separation of nucleic acids and proteins. Separation of large (macro) molecules depends upon 2 forces: charge and mass. When a biological sample, such as proteins or DNA, is mixed in a buffer solution and applied to a gel, these 2 forces act together. The electrical current from one electrode repels the molecules, while the other electrode simultaneously attracts the molecules. The frictional force of the gel material acts as a “molecular sieve,” separating the molecules by size. During electrophoresis, macromolecules are forced to move through the pores when the electrical current is applied. Their rate of migration through the electric field depends on the strength of the field, size, and shape of the molecules, relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving. After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other.
Agarose
There are 2 basic types of materials used to make gels: agarose and polyacrylamide. Agarose is a natural colloid extracted from seaweed. It is very fragile and easily destroyed by handling. Agarose gels have very large “pore” size and are used primarily to separate very large molecules, with a molecular mass greater than 200 kdal. Agarose gels can be processed faster than polyacrylamide gels, but their resolution is inferior. That is, the bands formed in the agarose gels are fuzzy and spread far apart. This is a result of pore size and cannot be controlled. Agarose is a linear polysaccharide (average molecular mass about 12,000) made up of the basic repeat unit agarobiose, which composes alternating units of galactose and 3, 6-anhydrogalactose. Agarose is usually used at concentrations between 1% and 3%. Agarose gels are formed by suspending dry agarose in an aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel.
Polyacrylamide
There are 2 basic types of materials used to make gels: agarose and polyacrylamide. The polyacrylamide gel electrophoresis (PAGE) technique was introduced by Raymond and Weintraub (1959). Polyacrylamide is the same material that is used for skin electrodes and in soft contact lenses. Polyacrylamide gel may be prepared so as to provide a wide variety of electrophoretic conditions. The pore size of the gel may be varied to produce different molecular sieving effects for separating proteins of different sizes. In this way, the percentage of polyacrylamide can be controlled in a given gel. By controlling the percentage (from 3% to 30%), precise pore sizes can be obtained, usually from 5 to 2000 kdal. This is the ideal range for gene sequencing, protein, polypeptide, and enzyme analysis. Polyacrylamide gels can be cast in a single percentage or with varying gradients. Gradient gels provide a continuous decrease in pore size from the top to the bottom of the gel, resulting in thin bands. Because of this banding effect, detailed genetic and molecular analysis can be performed on gradient polyacrylamide gels. Polyacrylamide gels offer greater flexibility and more sharply defined banding than agarose gels.
Mobility of
a molecule = (applied voltage) × (net charge of the molecule)/ friction
of
the molecule (in the electrical field)
V (velocity)
= E (voltage) × q (charge)/f (frictional coefficient).
Polyacrylamide Gel Electrophoresis
Polyacrylamide
is the solid support for electrophoresis when polypeptides, RNA, or DNA
fragments are analyzed. Acrylamide plus N, N’-methylene-bis-acrylamide in a
given percentage and ratio are polymerized in the presence of ammonium
persulfate and TEMED (N, N, N’, N’-tetra-methyl-ethylene-diamine) as catalysts.
Safety and Practical Points
Acrylamide and bis-acrylamide are toxic as long as they are not polymerized.
Buffer
(usually Tris) and other ingredients (detergents) are mixed with acrylamide
before polymerization.
Degassing of
acrylamide solution is necessary before pouring the gel because O2 is a strong
inhibitor of the polymerization reaction.
Polyacrylamide Gel Electrophoresis of
Proteins
Under nondenaturing conditions.
Under
denaturing conditions.
Isoelectric
focusing.
These
techniques are used to analyze certain properties of a protein such as:
isoelectric point, composition of a protein fraction or complex, purity of a
protein fraction, and size of a protein. We will concentrate on denaturing
polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
(SDS-PAGE) and a reducing agent (DTT, or dithioerithritol, DTE). The protein is
denatured by boiling in “sample buffer,” which contains:
1. Buffer pH
6.8 (Tris-HCl).
2. SDS.
3. Glycerol.
4. DTT or DTE.
5. Bromophenol
blue (tracking dye).
Discontinuous
Polyacrylamide Gel Electrophoresis
This type of
polyacrylamide gel consists of 2 parts:
1. The larger running (resolving) gel
2. The shorter upper stacking gel.
The running gel has a higher percentage (usually 10%–15%) of
acrylamide and a Tris-HCl buffer of pH 8.8.
The stacking gel usually contains 5% acrylamide and a
Tris-HCl buffer of pH 6.8.
The buffer used in SDS-PAGE is Tris-glycine with a pH of
about 8.3.
Determination of the
Molecular Weight of a Polypeptide by SDS-PAGE
Since all polypeptides are wrapped with SDS and thus are strongly negatively charged, they migrate through the running gel according to their size (small polypeptides migrate faster than large ones!). There is a linear relationship between the log of the molecular weight of the polypeptide and its migration during SDS-PAGE. Standard polypeptides have to be run on the same gel and a curve of their migration versus the log of their molecular weight has to be generated.
PREPARATION OF SDS-POLYACRYLAMIDE GELS
Materials
·
Casting
gel unit for electrophoresis
·
Siliconized
Pasteur pipettes
·
Syringes
equipped with blunt stub-nosed needles
·
Vacuum
chamber for degassing gels
·
Micropipettes
·
(10–300
mL) Stock 30%T:0.8%C acrylamide monomer
·
1.5
M Tris-HCl buffer, pH 8.8
·
10%
(w/v) SDS
·
10%
(w/v) ammonium persulfate
TEMED acrylamide is a powerful neurotoxin. Do not breathe
powder or otherwise come in contact with the monomer. Wear gloves at all times.
Separation gel mixed just prior to use
• 20 mL of acrylamide monomer
• 15 mL of Tris-HCl Buffer, pH 8.8
• 0.6 mL of 10% (w/v) SDS
• 24.1 mL of H2O.
Stacking gel mixed
just prior to use
• 2.66 mL of acrylamide monomer
• 5.0 mL of Tris buffer, pH 8.8
• 0.2 mL of 10% (w/v) SDS
• 12.2 mL of H2O
Procedure
1. Assemble your slab gel unit with the glass sandwich set in the casting mode with 1.5-mm spacers in place.
2. Prepare a separating gel from the ingredients listed.
3. Add the separating gel to a side arm flask, stopper the
flask, and attach to a vacuum pump equipped with a cold trap. Turn on the
vacuum and degas the solution for approximately 10 minutes. During this period,
gently swirl the solution in the flask.
4. Turn off the vacuum, open the flask, and add 200 mL of
ammonium persulfate and 20 mL of TEMED to the solution.
5. Add the stopper to the flask and degas for an additional 2
minutes while gently swirling the solution to mix the 2 accelerators. Use this
solution within a few minutes of mixing, or it will gel in the flask.
6. Transfer the degassed acrylamide solution to the casting
chamber with a Pasteur pipette. Gently fill the center of the glass chamber
with the solution by allowing the solution to run down the side of one of the
spacers. Be careful not to introduce air bubbles during this step.
7. Adjust the level of the gel in the chamber by inserting a
syringe equipped with a 22-gauge needle into the chamber and removing excess
gel.
8. Immediately water layers the gels to prevent formation of
a curved meniscus. Using a second syringe and needle, add approximately 0.5 mL
of water to the chamber by placing the tip of the needle at an angle to a
spacer and gently allowing the water to flow down the edge of the spacer and
over the gel. Add an additional 0.5 mL of water to the chamber by layering it
against the spacer on the opposite side of the chamber. Done appropriately, the
water will form a layer over the gel, and a clear line of demarcation will be
observed as the gel polymerizes.
9. After 30 minutes, the gel should be polymerized. If
degassing was insufficient, or the ammonium persulfate not fresh, the
polymerization may take an hour or more. When the gel is polymerized, lift the
gel in its casting chamber and tilt to decant the water layer.
10. Prepare a stacking gel from the listed ingredients.
11. Degas the stacking gel as in step 3.
12. Add 75 mL of ammonium persulfate and 10 mL of TEMED to
the stacking gel and degas for an additional 2 minutes.
13. Add approximately 1 mL of stacking gel to the gel chamber
and gently rock back and forth to wash the surface of the separating gel. Pour
off the still-liquid stacking gel and dispose of properly. Remember that liquid
acrylamide is extremely hazardous!
14. Add fresh stacking gel until it nearly fills the chamber,
but allow room for the insertion of a Teflon comb used to form sample wells.
Carefully insert a Teflon comb into the chamber. Adjust the volume of the
stacking gel as needed to completely fill the spaces in the comb. Be careful
not to trap any air bubbles beneath the combs. Oxygen inhibits polymerization,
and will subsequently result in poor protein separations.
15. Allow the gels to polymerize for at least 30 minutes
prior to use.
SEPARATION OF PROTEIN STANDARDS: SDS-PAGE
Materials
·
10%
SDS-polyacrylamide gel
·
Protein
standards
·
2X-SDS
sample buffer
·
1X-SDS
electrophoresis running buffer (Tris-Glycine + SDS)
·
0.001%
(w/v) bromophenol blue Micropipettes with flat tips for electrophoresis wells.
Procedure
1. Remove
the Teflon combs from the prepared gels by gently lifting the combs from the
chamber. Rinse the wells (formed by the removal of the combs) with distilled
water and drain it off.
2. Fill the
wells and the chamber with running buffer.
3. Prepare
aliquots of a known protein standard by mixing equal parts of the protein
standard with 2X sample buffer.
4. Using a
micropipette, add the sample to the bottom of a well. Add the blue to a
separate well.
5. Remove
the gel from its casting stand and assemble it into the appropriate slab unit
for running the electrophoresis. Be sure to follow the manufacturer’s
directions for assembly.
6. Pour a
sufficient quantity of running buffer into both the lower and upper chambers of
the electrophoresis apparatus until the bottom of the gel is immersed in
buffer, and the top is covered, while the electrodes reach into the buffer of
the upper chamber. Be careful not to disturb the samples in the wells when
adding buffer to the upper chamber.
7. Assemble
the top of the electrophoresis apparatus and connect the system to an
appropriate power source. Be sure that the cathode (+) is connected to the
upper buffer chamber.
8. Turn on
the power supply and run the gel at 20 mA constant current per 1.5 mm of gel.
For example, if 2 gels are run, each with 1.5-mm spacers, the current should be
adjusted to 40 mA. One gel with 1.5-mm spacers should be run at 20 mA, while a
gel with 0.75-mm spacers should be run at 10 mA.
9. When the
tracking dye reaches the separating gel layer, increase the current to 30 mA
per 1.5-mm gel.
10. Continue
applying the current until the tracking dye reaches the bottom of the
separating gel layer (approximately 4 hours).
11. Turn off
and disconnect the power supply. Disassemble the gel apparatus and remove the
glass sandwich containing the gel. Place the sandwich flat on paper towels and
carefully remove the clamps from the sandwich.
12. Working
on one side of the sandwich, carefully slide 1 of the spacers out from between
the 2 glass plates. Using the spacer or a plastic wedge as a lever, gently pry
the glass plates apart without damaging the gel contained within.
13. Lift the
bottom glass plate with the gel and transfer the gel to an appropriate
container filled with buffer, stain, or preservative. The gel may at this point
be used for Coomasie Blue staining, silver staining, enzyme detection, Western
blots, or more advanced procedures, such as electroblotting or electro elution.
If prestained protein standards were used, the gels may be scanned directly for
analysis. Place the gel into 50% methanol and gently rock the container for
about 30 minutes prior to scanning. This can be accomplished by placing the
gels into a flat dish and gently lifting the edge of the disk once every 30
seconds. There are commercially available rocker units for this purpose. If the
gel is to be dried, use a commercial gel dryer such as (SE 1160 Slab Gel
Dryer). Following the manufacturer’s directions demonstrates a dried and
stained gel containing a series of proteins of known molecular weights.
14. Plot the
relative mobility of each protein against the log of its molecular weight.
Relative mobility is the term used for the ratio of the distance the protein
has moved from its point of origin (the beginning of the separating gel)
relative to the distance the tracking dye has moved (the gel front). The ratio
is abbreviated as Rf. Molecular weight is expressed in daltons, and presents a
plot of the relative molecular weight of protein standards against the log of
their molecular weight.
COOMASSIE BLUE STAINING OF PROTEIN
GELS
·
Protein
gel
·
0.25%
(w/v) Coomassie Brilliant Blue R 250 in methanol-water-glacial acetic acid
(5/5/1), filtered immediately before use
·
7%
(v/v) acetic acid
·
Commercial
destaining unit (optional)
Procedure
1. Place a
gel (prepared as in Exercise 2) in at least 10 volumes of Coomassie Blue
staining solution for 2–4 hours. Rock gently to distribute the dye evenly over
the gel.
2. At the
conclusion of the staining, wash the gels with water a few times.
3. Place the
gels into a solution of 7% acetic acid for at least 1 hour.
4. If the
background is still deeply stained at the end of the hour, move the gels to
fresh 7% acetic acid as often as necessary. If a commercial destainer is
available, this will decrease the time required for stain removal. Follow the
manufacturer’s directions for use of the destainer.
5. Place the
gels into containers filled with 7% acetic acid as a final fixative.
6.
Photograph the gels or analyze the gels spectrophotometrically.
Notes
Coomassie
Brilliant Blue R 250 is the most commonly used staining procedure for the
detection of proteins. It is the method of choice if SDS is used in the
electrophoresis of proteins, and is sensitive for a range of 0.5 to 20
micrograms of protein. Within this range, it also follows the Beer-Lambert law
and, thus, can be quantitative as well as qualitative. The major drawback is
the length of time for the procedure and the requirement for destaining. Over staining
results in a significant retention of stain within the gel, and thus, a high
background stain, which might obliterate the bands. The length of time for
staining must be carefully monitored, and can range from 20 minutes to several
hours. If maximum sensitivity is desired, one should try 2 hours for a 5% gel
and 4 hours for a 10% gel. Destaining must be monitored visually and adjusted
accordingly.
SILVER STAINING OF
GELS
Materials
·
Protein
gel from
·
45%
(v/v) methanol + 12% (w/v) acetic acid.
·
5%
(v/v) methanol + 7% (w/v) acetic acid.
·
10%
Glutaraldehyde
·
0.01
M Dithiothreitol
·
Silver
nitrate solution Sodium citrate/formaldehyde
·
Kodak
Farmer’s Reducer or Kodak Rapid Fixer
Procedure
1. Fix gels
by gently rocking them in a solution of 45% methanol/12% acetic acid until the
gels are completely submerged. Fix for 30 minutes at room temperature. 2.
Remove the fixative and wash twice for 15 minutes each with 5% ethanol/ 7%
acetic acid. (Gels thicker than 1 mm require longer washing.)
3. Soak the
gels for 30 minutes in 10% glutaraldehyde.
4. Wash thrice with deionized water, 10
minutes each.
5. Place in
dithiothreitol for 30 minutes.
6. Place in
silver nitrate solution for 30 minutes.
7. Wash for
1 minute with deionized water. Dispose of used silver nitrate solution
immediately with continuous flushing. This solution is potentially explosive
when crystals form upon drying.
8. Place in
sodium citrate/formaldehyde solution for 1 minute.
9. Replace
the sodium carbonate/formaldehyde solution with a fresh batch, place gels on a
light box, and observe the development of the bands. Continue to rock gently as
the gel develops.
10. When the
desired degree of banding is observed (and before the entire gel turns black),
withdraw the citrate/formaldehyde solution and immediately add 1% glacial
acetic acid for 5 minutes.
11. Replace
the glacial acetic acid with Farmer’s reducer or Kodak Rapid Fixer for 1
minute. Remove Farmer’s reducer and wash with several changes of deionized
water.
12.
Photograph or scan the gel with a densitometer, which produces typical silver
stained protein gel.
13. For
storage, soak the gel in 3% glycerol for 5 minutes and dry between dialysis
membranes under reduced pressure at 80–82°C for 3 hours. Alternatively, place
the wet gel into a plastic container (a storage bag will do) and store at room
temperature. If desired, the gels may be dried between Whatman 3-MM filter
paper for autoradiography, or dried using a commercial gel dryer.
DOCUMENTATION
Materials
·
Polaroid
camera (Fotodyne Foto/Phoresis I or equivalent) or 35-mm camera equipped with macro lens
·
Stained
gel
Procedure
1.
Photograph the gels.
2. Use the
photographs or negatives to measure the distance from the point of protein
application (or for 2 gel systems, the line separating the stacking and
separating gels) to the final location of the tracking dye near the bottom of
the gel.
3. Measure
the distance from the point of origin to the center of each band appearing on
the gel.
4. Divide
each of the values obtained in step 3 by that obtained in step 2 to obtain the
relative mobility (the Rf value) for each band.
5. Using
either the graph of Rf values and molecular weights from Exercise 2, compute
the molecular weights of each band.
Optional
Scan the
negative with a densitometer and compute Rf values based on the distances from
the point of origin to the peak tracing for each protein band. Integration of
the area of each peak will yield quantitative data, as well as the molecular
weight.
 |
| SCHEME OF ELECTROPHORESIS |
No comments:
Post a Comment