Protein Electrophoresis

Safety Precautions:
Goggles, gloves and lab coats must be worn at all times during this experiment. Care
must be taken while working with TEMED, which is harmful if inhaled, swallowed, or absorbed
through the skin. TEMED is extremel destructive to tissue of the mucous membranes and upper
respirator tract, ees and skin. !D!, Coomassie brilliant blue, and bromophenol blue can also
irritate skin and ees. "mmonium persulfate is a strong oxidi#er and ma cause fire when in
contact with other materials. $t can cause burns to skin and ees, and ma cause skin allergies
and respirator reactions. Therefore, do not hold our face close to the bottles% open the bottles
under hood and avoid inhaling and contact with skin while handling these materials in the lab.
"crlamide monomer is a neurotoxin. Extreme caution must be used when working with the
non&polmeri#ed version of this compound. 'ear gloves at all times.
Introduction:
(rogress during protein purification is often monitored b sub)ecting column fractions to
an electric field and separating the proteins present based on their si#e. *nce separated, the
proteins can be visuali#ed b adding a de that binds to peptide bonds. The separation takes
place in a cross&linked gel&matrix of polacrlamide. The proteins can be denatured and
uniforml charged, as in !D!&polacrlamide gel electrophoresis +!D!&("GE,, or the ma be
in their native form, as in non&denaturing ("GE. !D!&("GE is the most widel used method for
-ualitativel anal#ing protein mixtures.
Cross&linked polacrlamide gels are formed from the polmeri#ation of acrlamide
monomer. The addition of N,N.&methlene&bis&acrlamide allows separate polacrlamide
chains to become cross&linked, forming a matrix of polmeri#ed polacrlamide of well&defined
structure and pore si#e. / varing the amounts of acrlamide and bis&acrlamide the extent of
polmeri#ation and cross&linking can be varied. The reaction is initiated b the addition of
ammonium persulfate and 0,0,0.,0.&tetramethlenediamine +TEMED, in an example of free
radical chemistr. The sulfate free radicals that form interact with the acrlamide monomers,
forming long strands of acrlamide polmers. These polmers are cross&linked b the bis&
acrlamide present in the solution. The radical reaction is -uenched b the presence of oxgen.
This laer of acrlamide is called the separating or resolving gel and usuall contains between 12
and 134 acrlamide.
(rotein samples are added to a second laer of acrlamide stacked on top of the resolving
gel. This laer is called the stacking gel, since it serves to concentrate the protein in the sample
into a tight band. This gel has a low percentage of acrlamide, usuall around 54, that contains
large pores which do not impede protein movement. Three ions present in the stacking gel,
protein, Cl
&
, and glcine, stack under the effect of the electric field according to their
electrophoretic mobilit. The Cl
&
ions migrate at the greatest speed, glcinate ions migrate at the
slowest speed, and the protein ions are tightl packed between the two ions. 6pon reaching the
resolving gel, the p7 changes from 8.9 to 9.9 and the glcinate ions pass the proteins and catch
up with the Cl
&
ions. This leaves the proteins to electrophorese at their own rates.
:or !D!&("GE, protein samples are boiled in buffer with &mercaptoethanol +/ME, and
!D!. /ME is present to reduce an disulfide bonds in the protein, while !D! is a negativel
charged sulfate derivate that binds to proteins. !D! binds at a rate of 1 !D! molecule per ;
amino acid residues and coats the protein with a uniform negative charge. 6nder these
conditions the protein becomes denatured, rod shaped, and highl charged. "ll proteins in the
mixture have the same shape and the same mass&to&charge ratio and the therefore separate
based on the molecular sieving effect of the gel.
!D!&("GE separates denatured proteins. $n some cases it is necessar to anal#e native
proteins, especiall if biological activit is to be assessed. $n nondenaturing gel electrophoresis,
the proteins are not denatured nor are the coated with !D!. The are loaded into the gel as is,
and since most proteins contain a net negative charge, the will migrate toward the anode.
Electrophoresis under these conditions depends on man parameters, including si#e, charge, and
shape.
The Experiment:
Materials for !D!&("GE +"<=E"D> M"DE,
1. !tock acrlamide solution? @24 acrlamide, 2.94 bis&acrlamide. Acrylamide
monomer is a neurotoxin and must be handled with care!
;. /uffers?
a. 1.3 M Tris&Cl, p7 9.9
b. 2.3 M Tris&Cl, p7 8.9
@. 124 ammonium persulfate, made fresh
5. 124 !D!
3. TEMED
8. Electrophoresis buffer? Tris +1;g,, glcine +3A.8g,, and !D! +;.2g,. "dd water to ; <
final volume, no p7 ad)ustment is necessar.
A. !ample /uffer +3B,?
a. 2.3 M Tris&Cl, p7 8.9 3.2 m<
b. !D! 2.3 g
c. !ucrose 3.2 g
d. &mercaptoethanol 2.;3 m<
e. /romophenol blue, 2.34 stock 3.2 m<
"dd water to a total volume of 32 m<
9. (rotein stain? 2.14 coomassie brilliant blue =;32 in 324 methanol, 124 glacial acetic
acid
C. Destain? 124 methanol, 124 glacial acetic acid
(rocedure
1. Clean glass plates with soap or ethanol. (repare plates for pouring of the gel as
illustrated b the instructor. Mark the plates at a point D; cm below the bottom of the
comb. >ou will add the resolving gel up to this mark.
;. "dd ;4 agarose to the chamber directl below the plates until it reaches the bottom of
the plates. "llow this agarose plug to cool and solidif while ou move to step @.
@. Mix the following in a 13 m< falcon tube +this should be enough acrlamide for two
gels,?
a. 1.3 M Tris&Cl, p7 9.9 ;.2 m<
b. 'ater ;.93 m<
c. !tock acrlamide 3 m<
d. 124 !D! 2.1 m<
e. "mmonium persulfate 2.23 m<
5. "dd A < of TEMED, gentl swirl the tube to ensure even mixing.
3. 6sing a (asteur pipet, transfer the separating gel mixture to the plates. "dd mixture until
it reaches the markings ou made on the plates earlier.
8. "dd ethanol to the top of the gel to protect the polmeri#ing acrlamide from oxgen and
allow the gel to set until it has full polmeri#ed.
A. (repare our samples b mixing 12E< of each of our fractions with ;E< of the de, and
mixing 12E< of the protein mix with ;E< of the de. Take 3E< of the molecular markers
and use them as is.
9. (repare the stacking gel +this should be enough for two gels,?
a. 2.3 M Tris&Cl, p7 8.9 2.3 m<
b. !tock acrlamide 2.8A3 m<
c. 'ater @.A3 m<
d. 124 !D! 2.23 m<
e. "mmonium persulfate 2.2;3 m<
C. =emove the ethanol laer from the top of the polmeri#ed gel b decanting over the sink
and using kimwipes to remove as much ethanol as possible. (lace the comb into position.
12. "dd A < of TEMED to the stacking gel and mix gentl. <aer this solution on top of the
separating gel, filling up all the wa to the top of the glass plates. Make sure there are no
bubbles below the teeth of the comb.
11. /oil our samples prepared above for ; minutes. D* 0*T /*$< T7E M*<EC6<"=
M"=FE=!.
1;. *nce the stacking gel has set, mark the positions of the wells with a marker, remove the
comb from the plates and place the gels in the buffer tank.
1@. /oil our protein samples for two minutes. 6se 9 ul of protein and ; ul of loading de.
15. :ill the reservoir tank and the space between the plates until the edge of the inner plate
and the tank lower electrodes are covered with electrophoresis buffer. Then load the
samples into the wells.
13. =un the gel at a constant current of ;3 m" per gel until the blue de is at the bottom of
the gel.
18. !top the gel and empt out the buffer. =emove the gel plates from the electrophoresis
apparatus. 6se the spacer to separate the two plates, detach and discard the stacking gel
on the top, place a notch on one side of the gel to mark which sample was loaded at
which side, and finall separate the gel from the plate. !tain the gel for D@2 min.
1A. Decant the stain into the waste )ar in the hood and begin to destain until the protein bands
are clear.