SDS_PAGE_WB

7/21/2019 SDS_PAGE_WB

http://slidepdf.com/reader/full/sdspagewb 1/6

SDS-PAGE and Western BlottingAdapted from Horwitz lab procedures

Ref: Amersham Life Science' s booklet on ECL- estern blottin! protocols"

I. SDS-PAGE:

Ref: BIO-RAD ' s " Mini-PROTEAN 3 Cell Asseml! G"ide "

1. Assemble gel sandwicha.Place together short and spacer plates so that both places are flush at bottom on a level surface. b.Slide the 2 plates into casting frame with the smaller plate-facing front. oc! pressure cams to

secure the glass plates.

c.Place a gra rubber gas!et on bottom of gel assembl apparatus.

d.#nsert gel cassette sandwich into gel apparatus$ Place sandwich on top of gra casting stand

gas!et and engage the top of the spacer plate with the spring loaded lever. %a!e sure the lever

pushes the spacer plate down so both plates are perpendicular against the gra rubber gas!et.

2. Prepare the separating gel solution.

a. &or each gel( (..(1)m1 of separating gel is needed.* Swirl gentl after APS addition( then use a

Pasteur pipette to insert solution between glass plates up to the bottom of the green bar.

b.#mmediatel after adding separating gel( use another Pasteur pipette to add butanol on top of

gel. &#f using a mi+ture of butanol$ water( ma!e sure onl to withdraw the butanol on the toplaer.*

c.Allow separating gel to polmeri,e. est b suee,ing the top of the used Pasteur pipette &for

separating gel*$ if solution has solidified( polmeri,ation has occurred.

d.#n the meantime( prepare the stac!ing gel. &or each gel( (..(/m1 of stac!ing is needed. *

e.0hen separating gel has polmeri,ed( pour out and wash out butanol in sin! with tap water.

3. Stac!ing gel

a.se a Pasteur pipette to la on stac!ing gel on top of polmeri,ed separating gel. et it flowover the top. #nsert a 1./mm( 1)-well comb between the glasses while ma!ing sure no air

bubbles are trapped between the teeth of the comb.

b.#n the meantime( prepare the gel electrophoresis apparatus that holds # running buffer.

c.0hen the stac!ing gel has polmeri,ed( carefull remove comb. inse and irrigate the wells

with # running buffer thoroughl.4. emove gel plates from their green cases( then place plates in running buffer apparatus with the

smaller plate facing inward.

/. ill he case full with running buffer. ill the middle section until it overflows into 2 side sections.

5. ill the wells with samples$

a.ill 2 ends with /u %0 mar!ers.

b.ill rest of 6 wells with 4)ul of prepared specific sample solution.

c.ill each well slowl -the goal is to get all of the sample solution into each well. r to avoid

bubbles in wells that ma cause overflow of solution into other wells

d.0hen wor!ing with 2 gel plates( ma!e sure to label on the outside of the container which

protein it is to be used for.

7. %atch the 8 and -electrode ends( and set the apparatus to run lhr9lhr1/min : 13);.

6. After lh 1/min( chec! to ma!e sure the blue band has been run all the wa to the bottom of thegel plate.

<. ransfer apparatus

a.Prepare 1 of transfer buffer

b.Set up gel cassette

i. =lear side down

ii. iber pad$ -soa! and suee,e out air bubbles in transfer buffer

iii. ilter paper$ -pre-wet in transfer buffer

iv. %embrane$ -briefl soa! in methanol( then la in dd>2)



v. ?el- to be placed in the ne+t step

vi. emove gel case from apparatus. =rac! plates open with a wet ra,or and gentl slide

stac!ing gel off

vii. Place gel on-top of membrane paper using a wet ra,or

viii. ilter paper$ - &pre-wet in transfer buffer*i+. hen fiber pad &air bubbles soa!ed and suee,ed out in transfer buffer*.

c.=lose together cassette and insert into transfer buffer apparatus -blac! side facing blac! side.1). Set up running buffer to run for 1 hour : 1));.

11. Put in the ice bloc!.

II. Western Blotting

1. emove membrane paper from # transfer buffer apparatus. &ememberwhich side the gel proteins transferred onto( and sign our name that side's bottom right handcorner.*

2. %a!e @lotto &2/m for one blot in a /)m test tube.* Place tube onvorte+ at highest speed for a few seconds.

a./ wBv$ -e+. )./gB1)m or 1.2/gB2/m

3. Place membrane in @lotto for 1 hr : wBconstant agitation.4. 0ash blot 3/min in @S-.

/. Place membrane in primar solution &1)m for 1 blot* : for 1 hr. or overnight : 4 =wBconstant agitation.

5. Pour primar solution bac! into its original test tube for reuse.

7. 0ash blot 3/min in @S-.

6. #n the meantime( prepare secondar antibod solution- 1$1)))) dilution ofmonoclona1Bpolclonal antibod in @lotto &1)m for one blot*.

<. Place membrane in secondar solution : for 1 hr. or overnight : 4 = wBconstant agitation.

1). 0ash blot 3/min in @S-.

11. Prepare detection reagent$ 1m reagent A 8 1m reagent @

12. Place blot on Saran wrap. Pipette above reagent onto blot( covering the entire blot surface.

13. et sit for 1min.a. 0hen developing 2 blots at the same time( !eep the 2nd one

wrapped in @S-.

14. emove e+cess liuid with filter paper.

1/. Cevelop blot.

15. #f needed( store the blot wrapped in Saran wrap with @S- at 4=.

III. Stripping & Reprobing

1. Stripping

a. 92/m guanidine >= for 1)min : wBconstant agitation.

b. 0ash blot 1/min in @S-.

2. @loc! with @lottoD&?E E step 2 from above*



S#S-$A%E and estern blottin! buffers

& sample buffer ).52/ ml 1 % ris( p> 5.6 &12/ m%*

&/ ml* 1 ml 2) SCS &4( or ).2 g*

Store : -7) °= 1 ml glcerol &2)*in 1 ml aliuots ).1 ml 1 bromphenol blue in 1) FtE> &).)2*

&Add 2)) m% C G ).1/42 g or 1) @%F if needed for reducing conditions*

ill to / ml with >2E &92.27/ ml*

( sample buffer )./ ml 2./ % ris( p> 5.6 &2/) m%*

&/ ml* 2 ml 2) SCS &6*

Store : -7) °= 2 ml glcerol &4)*

in 1 ml aliuots ).2 ml 1 bromphenol blue in 1) FtE> &).)4*&Add 4)) m% C G ).3)64 g if desiredH itIs not recommended to add 2) @%F to the

4 buffer*

ill to / ml with >2E &9 ).3 ml*

)* runnin! buffer 2< g ris base &24) m%*

Store : 144 g glcine &1.< %*

1) g SCS

;olume to 1 &donIt p>*

+ransfer buffer 2.< g ris base &12 m%*

%a!e fresh or 14.4 g glcine &1)) m%*

Store : 2)) ml %eE>wBo %eE> 1 ml 2) SCS

;olume to 2 &donIt p>*

Separatin! !els ml Protogel & G desired final*&3) ml* 7.6 ml Protogel buffer

3) ul F%FC

;olume to 3) ml with >2ECegas if desired( then add$

3)) ul freshl-made 1) APS in >2E

Stackin! !els 1.3 ml Protogel &4 final*

&1) ml* 2./ ml Protogel stac!ing buffer 5.1 ml >2E

1) ul F%FC

/ ul 2)))+ phenol red concentrate &Sigma*

Cegas if desired( then add$/) ul 1) APS

Protogel &Jational Ciagnostics* contains 3) wBv acrlamide( ).6 wBv bis-acrlamide

Protogel buffer contains 1./ % ris->=l( ).364 SCS( p> 6.6Protogel stac!ing buffer contains ).12/ % ris->=l( ).1 SCS( p> 5.6



Coomassie blue stainin! solution ).1 wBv =oomassie blue 2/)

4) %eE>

1) >Ac

#estainin! solution 3) %eE>

1) >Ac

Lon!er destainin! solution / %eE>7./ >Ac

/ glcerol

+,S-+ween 4) ml 1 % ris->=l( p> 7./ &2) m%*Stable for wee!s /4.6 ml / % Ja=l &137 m%*

: &ween ;olume to 2 with >2E( then add$

can o+idi,e* 2 ml ween &).1( isher ScientificKfor @S Lust omit this*

&can add ).)2 JaJ3 to prevent bacterial growth*

Strippin! buffer 3 ml 2) SCS &2*

).7/ ml 2./ % ris( p> 5.6 &52./ m%*

).21 ml @%FKdG1.114( 14.3 % stoc! liuid &1)) m%*

25.)4 ml >2E

)* $,S 6) g Ja=l

2 g M=l14.4 g Ja2>PE4

2.4 g Ja>2PE4

Add <)) ml >2E

p> to 97.) with JaE> pellets;olume to 1 and filter

)* +AE 24.2 g ris base

/.71 ml >Ac1) ml )./ % FCA( p> 6.)

;olume to /)) ml with >2E

) ,SA blockin! and Ab dilution buffer 1 @SA in @S- or @S 8 ).)1-).1 riton



Lsis buffer .)* ml/

inal concentrations in the lsis buffer are in parentheses.

Sol"tions stored at #° C

/.55/ ml dd>2E

1 ml )./ % β-glcerophosphate( p> 7.3 &/) m%* NN)./ %$ 1.46/ g of hdrate in 1) ml >2E

1 ml ).1 % JaPP &1) m%* NN).1 % JaPP$ ).6<22 g in 2) ml >2E).5)) ml )./) % Ja &3) m%* NN)./ % Ja$ ).41<< g in 2) ml >2E

)./)) ml 1 % ris( p> 7./ &/) m%* NN1 % ris$ 12.11 g ris base in 1)) ml >2E

)./)) ml 2) riton -1)) &1* NN2) g riton -1)) &d G 1.)7* in 1)) ml >2E

).3)) ml / % Ja=l &1/) m%* NN/ % Ja=l$ 2.<2 g Ja=l in 1) ml >2E).1)) ml 1)) m% ben,amidine &1 m%* NN1)) m%$ ).313 g in 2) ml >2E

).)4) ml /)) m% F?A &2 m%* NN/)) m% F?A$ 3.6)4 g in 2) ml >2EKincrease p>

Sol"tions stored at -$%° C

).)2/ ml 4) m% sodium orthovanadate &1)) µ%* NN4) m%$ ).)73/5 g in 1) ml >2E

).)2) ml /)) m% C &1 m%* NN/)) m% C$ ).771g in 1) ml >2E

Sensiti&e Items

-$%° C

).)2) ml / mgBml aprotinin &1) µgBml*

).)2) ml / mgBml leupeptin &1) µgBml*

).)1) ml 1 mgBml pepstatin &1 µgBml* NN2 mg in 2 ml %eE>

).)1) ml 1 mgBml %icrocstin &1 µgBml* made in FtE>

#° C

).2)) ml /) m% P%S &1 m%* NNmade in FtE>

,lockin! ,uffer

1 @SA in$

)./)) ml 1 % ris( p> 7./ &/) m%*

).3)) ml / % Ja=l &1/) m%*

).)2/ ml 2) riton -1)) &).)/*<.17/ ml dd>2E

ash ,uffer

)./)) ml 1 % ris( p> 7./ &/) m%*).3)) ml / % Ja=l &1/) m%*

<.2)) ml dd>2E

H0$1(

67 µl 6/ >3PE4 &stoc!* in 1) ml dd>2E.



2inase Assa ,uffer

Component Stock Conc" orkin! Conc" 3nits 4olume . l/ to add

to make )*** l

5otes

Stored at #° C

ris( p> 7./ 1))) 2) m% 3)

%g=l2 1))) 1/ m% 22./β-gl /)) / m% 1/

F?A /)) 1 m% 3

Stored at -$%° C

Ja3;E4 4) ).2 m% 7./C /)) ).2 m% ).5 Σ volume G 76.5 µl

PMA inhibitor 4/) ).4 µ% 2.57

PM= inhibitor 4/) 4 µ% 25.57

=almida,olium 1)))) 4 µ% 1.2 Cissolved in C%SE

APO 1))) 2/ µ% 7/ Cilute onl when read to

useP32-APOO )./// ).))/5 µ% 3) =old$>ot AP ratio G

4/)4./)Specific Activit of >ot G

3))) =iBmmolSpecific Activit of otal

AP G ).57 =iBmmol G

1.46F5 dpmBnmol

dd>2E 76/.67

O irst dilute the 1) m% stoc! b 1)-fold in water( then use 7/ µl of the diluted stoc!.

OO Cilute the stoc! &3.33 µ%( 1) µ=iBµl* 32P-AP b 5-fold in 1) m% &p> G 7.5* ricine and store in aliuots. Co

not free,eBthaw aliuots more than 3-4 times.

2inase ash ,uffer

Component Stock Conc" orkin! Conc" 3nits 4olume .ml/ to add to

make )* ml

5otes

ris( p> 7./ 1))) 2) m% ).3

%g=l2 1))) 1/ m% ).22/

β-gl /)) / m% ).1/

F?A /)) 1 m% ).)3

Ja3;E4 4) ).2 m% ).)7/

C /)) ).2 m% ).))5 Σ volume G ).765 mlK

ta!e 76.5 µl out for

MA@H then add onl

6.2<25 ml dd>2E

dd>2E <.214

eaction mi+$ 2) µl M0@

2) µl MA@

2) µl %@P &stoc! concentration G 2 mgBml*

otal radioactivit per well G 1 µ=i &from /) µ=i in 1))) µl of MA@*

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SDS_PAGE_WB