SDS- PAGE Technique.doc

8/21/2019 SDS- PAGE Technique.doc

http://slidepdf.com/reader/full/sds-page-techniquedoc 1/6

SDS-PAGE

Principle

Electrophoresis is the study of the movement of charged molecules in an electric field. The

generally used support medium is cellulose or thin gels made up of either polyacrylamide or

agarose. Cellulose is used as support medium for low molecular weight biochemicals such as

amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for

larger molecules like proteins. The general electrophoresis techniques cannot be used to

measure the molecular weight of the biological molecules because the mobility of a substance

in the gel is influenced by both charge and size. In order to overcome this, if the biological

samples are treated so that they have a uniform charge, electrophoretic mobility then depends

primarily on size. The molecular weight of protein maybe estimated if they are subected to

electrophoresis in the presence of a detergent sodium dodecyl sulfate !"#"$ and a reducing

agent mercaptoethanol !β %E$. "#" disrupts the secondary, tertiary and quaternary structure

of the protein to produce a linear polypeptide chain coated with negatively charged "#"

molecules. &ll proteins are now negatively charged with similar charge density and thus can

be separated on the basis of their size only. '.(grams of "#" binds per gram of protein.

%ercaptoethanol assists the protein denaturation by reducing all disulfide bonds.

"#")*&+E is used mainly for the following purpose

a. Estimation of protein size.

b. #etermination of protein subunits or aggregation structures.

c. Estimation of protein purity.

d. *rotein quantitation.

e. %onitoring protein integrity.

f. Comparison of the polypeptide composition of different samples.

g. &nalysis of the number and size of polypeptide subunits.

h. *ost)electrophoresis applications, such as -estern blotting.

Procedure:

1. Gel Cassette Sandwich Preparation (Demonstration)

'. +lass Cassette and Casting "tand &ssembly

Note: Ensure the casting stand, casting frames, and glass plates are clean and dry before setting up

the casting stand assembly. During regular use, a powder residue may build up behind the pressure

cams of the casting frame at the pivot point. This powder should be removed before each use.

a. *lace the Casting rame upright with the pressure cams in the open position and facing

forward on a flat surface.b. "elect a "pacer *late of the desired gel thickness and place a "hort *late on top of it

Note:1. Gloves should be worn at all times while performing D!"#GE.$. To insure proper alignment and casting, the glass plates, spacers, combs and casting stand gas%ets must be clean and dry. The glass plates should be cleaned with &'( ethanol.



c. /rient the "pacer *late so that the labeling is 0up0. "lide the two glass plates into the

Casting rame, keeping the "hort *late facing the front of the frame !side with pressure

cams$

Note: Ensure both plates are flush on a level surface and labeling on the pacer "late is oriented

correctly. )ea%ing may occur if the plates are misaligned or oriented incorrectly.

d. -hen the glass plates are in place, engage the pressure cams to secure the glass cassette

sandwich in the Casting rame. Check that both plates are flush at the bottom. Engage the

spring loaded lever and place the gel cassette assembly on the gray casting stand gasket.

Insure the horizontal ribs on the back of the Casting rame are flush against the face of the

Casting "tand and the glass plates are perpendicular to the level surface. The lever pushes

the "pacer *lace down against the gray rubber gasket.

e. 1epeat steps a2d for a second gel.

Figure 1. Mini-PR!EA" # s$stem components.

%. &asting t'e gels (Demonstration)

*repare '3.4 5 resolving6separating gel and (.75 stacking gel. *lease refer to appendi8 ' for

the recipe.

'. *repare the separating gel monomer solution by combining all reagents e8cept

ammonium persulfate !&*"$ and TE%E#. #eaerate and mi8 the solution after adding each

reagent by swirling the container gently.

3. *lace a comb completely into the assembled gel sandwich. -ith a marker pen, place a

mark on the glass plate ' cm below the teeth of the comb. This will be the level to which the

separating gel is poured. 1emove the comb.



9. &dd &*" and TE%E# to the monomer solution and mi8 well by swirling gently. *ipette the

solution to the mark.

(. Immediately overlay the monomer solution with ' ml. of water. :se a steady, even rate of

delivery to prevent mi8ing with the gel.

7. &llow the gel to polymerize for (7 minutes to ' hour. *our the water overlaying the gel anddrain the e8cess water with strips of filter paper.

4. *repare the stacing gel monomer solution. Combine all reagents e8cept &*" and

TE%E#. #eaerate and mi8 the solution by swirling gently.

;. *lace a comb in the gel sandwich.

<. &dd &*" and TE%E# to the solution and pipette the solution down one of the spacer until

the sandwich is filled completely.

=. &llow the gel to polymerize for '7 minutes.

#. Preparation o samples (*$ tutor)The clone was grown for (hours and induced using I*T+ for ne8t four hours. The culture was

pelleted and resuspended in samples buffer. *lease refer to appendi8 ' for the recipe.

+. Mini-PR!EA" # Electrop'oresis Module Assem,l$ (Demonstration)

'. 1emove the +el Cassette &ssemblies from the Casting "tand. 1otate the cams of the

Casting rames inward to release the +el Cassette "andwich

3. *lace a +el Cassette "andwich into the slots at the bottom of each side of the Electrode

&ssembly. >e sure the "hort *late of the +el Cassette "andwich faces inward toward the

notches of the :)shaped gaskets9. ?ift the +el Cassette "andwich into place against the green gaskets and slide into the

Clamping rame

(. *ress down on the Electrode &ssembly while closing the two cam levers of the Clamping

rame to form the Inner Chamber and to insure a proper seal of the short plate against the

notch on the :)shaped gasket. "hort plate must align with notch in gasket.

Note: Gently pressing the top of the Electrode #ssembly while closing the *lamping +rame cams

forces the top of the hort "late on each Gel *assette andwich to seat against the rubber gas%et

properly and prevents lea%ing.

7. ?ower the Inner Chamber &ssembly into the %ini Tank. ill the inner chamber with @'37 ml

of running buffer until the level reaches halfway between the tops of the taller and shorter

glass plates of the +el Cassettes.

Note: Do not overfill the nner *hamber #ssembly. E-cess buffer will cause the siphoning of buffer into

the lower chamber which can result in buffer loss and interruption of electrophoresis.

4. &dd @3AA ml of running buffer to the %ini Tank !lower buffer chamber$.

. oading t'e samples (*$ students)

'. 1inse the well a few times with running gel buffer using the syringe. ?oad the samples into

the wells with a pipette using gel loading tips. #emonstrators will load the first well with ?%-

!; Bl of ?%-$. Insert the tip !'A Bl$ to about ')3 mm from the well bottom before delivery.



3. ?oad the second and other well with 3A Bl of protein sample as described above. #o not

pipette the pellet at the bottom of the microfuge tube.

Note: )oad samples slowly to allow them to settle evenly on the bottom of the well. e careful not to

puncture the bottom of the well with the syringe needle or pipette tip.

/. Running t'e gel

'. Check that the buffer in the upper buffer chamber are full because leakage of the buffer

may occur.

3. *lace the lid on top of the lower buffer chamber. %ake sure that the connection is correct,

ie. black to black and red to red.

9. &ttach the electrical leads to a suitable power pack with the proper polarity !black to black

and red to red$. 1un the gel at a constant current of 9A m&.

(. "top the electrophoresis when the tracker dye is @ ' cm above the end of the glass plates.

0. Remoing and staining t'e gel (*$ tutor)

Gel Remoal

'. &fter electrophoresis is complete, turn off the power supply and disconnect the electrical

leads.

3. 1emove the tank lid and carefully lift out the Inner Chamber &ssembly. *our off and discard

the running buffer.

ote &lways pour off the buffer before opening the cams to avoid spilling the buffer.

9. /pen the cams of the Clamping rame. *ull the Electrode &ssembly out of the Clamping

rame and remove the +el Cassette "andwiches.(. 1emove the gels from the +el Cassette "andwich by gently separating the two plates of

the gel cassette. The green, wedge shaped, plastic +el 1eleaser may be used to help pry the

glass plates apart.

ote To remove the gel from a 1eady +el Cassette, first slice the tape along the sides of the

1eady +el Cassette where the inner glass plate meets the outer plastic plate.

7. 1un the sharp edge of the +el 1eleaser or a razor blade along each spacer to separate the

gel from the spacer. 1emove the gel by floating it off the glass plate by inverting the gel and

plate under fi8ative or transfer solution, agitating gently until the gel separates from the plate.

4. 1inse the %ini)*1/TE& 9 cell electrode assembly, Clamping rame and %ini Tank withdistilled, deionized water after use.

Gel staining

(. *lace the removed gel in the container containing the Coomassie blue stain. %ake sure

that the gel is fully submerged in the staining solution.

7. "tain the gel for ' hour, agitate it slowly on a shaker.

4. #estain the gel in a destaining solution a few times until protein bands are visualized.

;. &ppro8imately determine the molecular weight of the visualized protein bands by comparing

them with the molecular weight markers.



Appendi2

Preparation o a 1%./ 3 Resoling4separating gel. +. ml4minigel

9A5 acrylamide D A.<5 >is)acrylamide '.<= ml

'.7 % Tris)Cl, p <.<, '.'37 ml

#istilled water A.77 ml'A5 ammonium persulfate !&*"$ 37 ml

'A5 "#" (7 ml

TE%E# 9 ml

7A5 +lycerol

Preparation o a +.3 stacing gel5 6.7 ml

9A5 &crylamide D A.<5 >is)acrylamide A.'( ml

A.7%Tris)Cl, p 4.< A.3 ml

#istilled water A.(7 ml

'A5 "#" ( ml &mmonium persulfate 3A ml

TE%E# ' ml

8se t'e ollo9ing guide to determine 3 acr$lamide in resoling gel:

75 for "#")denatured proteins 4A to 3AA k#a

;.75 for proteins 7A to '3A k#a

'A5 for proteins '4 to ;A k#a

'3.75 for proteins '7 to 4A k#a

'75 for proteins '3 to (7 k#a

The 5 acrylamide in the stacking gel is constant.

Molecular eig't Marers

*age1uler *restained *rotein ?adder !ermentas catF "%A(9'$ and find this to be a good

broad)range marker for proteins '( to ''4 k#a. ?oad 7 µl per lane for 'A)well comb.

&oomassie Stain (1 )

A.'5 coomassie 1)37A

(A5 ethanol

'A5 acetic acid

To (<A ml water add (3A ml =75 ethanol and 'AA ml acetic acid while stirring. &dd '.A g

coomassie 1)37A. Transfer to storage bottle that has been rinsed to remove any precipitate./

Sample *uer

Combine the following in a '7 ml conical tube and gently agitate on rotator until components

are dissolved. or 'A ml

; ml A.4 % Tris)Cl p 4.<

9 ml glycerol

' g "#"

'.3 mg bromophenol blue

16

Running *uer

4A.4 g Tris base3<< g +lycine



#issolve in '4AA ml water and bring the volume to 3 litres. Transfer to a 3 ? bottle using a

funnel. Carefully add %6 g SDS while stirring. &dding the "#" before transfer to the bottle will

result in bubbles, making transfer impossible. #ilute 7A ml of this stock with (7A ml water for

each electrophoresis run and mi8 thoroughly.

Documents

SDS- PAGE Technique.doc