SDS-PAGE: Pouring the resolving gel

1. Clean glass plates with soap and water, then with ethanol. Assemble the Glass plates and spacers. 2. For large gels, seal with 1 mL of resolving gel. Remove 2 mL and when sealant is polymeri ed, the remaining 1 mL of gel will be polymeri ed. Add to 2 ml of resolving gel, ! ml "#$#% and &' mL 1'( A)* to polymeri e sealant. *eal the large gel plates by po+ring 1 mL of resolving gel ,after adding the A)*-"#$#%. into the plates and tilting them to spread the sealant. Allow most of e/cess sealant to r+n o+t, b+t save a small amo+nt of e/cess to sit on the bottom to ma0e s+re it seals 1.2. 3hile this is going on, degas ,for 1' min+tes. the remaining 4+antity of +npolymeri ed resolving gel. For the minigel, attach the glass plate assembly to the apparat+s and place the whole gimisch in a small, flat "+pperware container. 3ith a )aste+r pipette, seal the bottom of the plates by spreading 1( agarose in *%* )AG# electrophoresis b+ffer along the bottom of the glass plates so it goes +p the crac0 by capillary action. 5. "ransfer the degased resolving gel to a bea0er and add A)*-"#$#%. $i/. 6+ic0ly add the resolving gel sol+tion to the center of the plates to a height abo+t ! cm from the top for the large plates. For the minigel, add the resolving gel with a )aste+r pipette. !. 6+ic0ly add isob+tanol to the top of this +ntil the level reaches the top of the plates. 7sob+tanol will prevent o/ygen from getting into the gel which co+ld o/idi e it and inhibit polymeri ation. 8. "he resolving gel sho+ld polymeri e in 5' min+tes. &. Add the stac0ing gel reagents to a flas0 and degas for ten min+tes. Pouring the stacking gel 9. )o+r off isob+tanol. )o+r water several times into the gel plate space to rinse off all the isob+tanol. %ry the watery interplate s+rface with a piece of 3hatmann paper.

:. "o polymeri e the stac0ing gel, adds A)*-"#$#%, mi/, and then po+r on top of the polymeri ed resolving gel. ;. 7nsert the comb straight on down, then po+r a little more stac0ing gel on the sides of the comb to f+lly seal it. Remove any b+bbles from +nderneath the comb, if possible, by moving the comb gently from side to side so the b+bbles get into the space in between and float +p. 1'. "he stac0ing gel sho+ld polymeri e in 2' to 5' min+tes. Load the gel 11. Attach the large gel plates containing the polymeri ed gel to the apparat+s via the clips provided. ,For the minigel, the plates are attached to the apparat+s when the gel is po+red.. 12. )o+r "ris<glycine electrophoresis b+ffer into the +pper and lower chambers. "he large gel ta0es abo+t 1 liter, the minigel ta0es abo+t 2'' ml. 15. Remove b+bbles trapped at the bottom of the glass plates in the large gel with a syringe. 1!. Fl+sh the wells with a syringe =+st before loading to get rid of any +npolymeri ed polyacrylamide that may seep in. 18. 3hen loading the gel, load something ,1> loading b+ffer in blan0 lanes. in every lane and the dye front will migrate more evenly. 1&. R+n large thin gels at a constant c+rrent of 2' mA. After the dye front enters the resolving gel, yo+ can t+rn the c+rrent +p to 5' mA. 3here yo+ stop the gel will depend on how big the smallest protein is that yo+ want to vis+ali e. 7f yo+ wait +ntil the dye front =+st flows o+t of the gel, it sho+ld ta0e abo+t 2 1-2 < 5 ho+rs for a large, thin gel to r+n. A thic0 gel co+ld ta0e & < 9 ho+rs. R+n minigels at 18 mA. 7t sho+ld ta0e abo+t an ho+r to r+n.

Western blotting
Transferring the gel 1. 3hile the gel is r+nning, prepare the transfer b+ffer, and c+t o+t a piece of nitrocell+lose and fo+r pieces of 3hatmann filter paper for the transfer.

1+r ?iorad tan0 ta0es abo+t 5 liters of transfer b+ffer. 2. C+t o+t the 3hatmann filter papers so they each are slightly larger than the gel in each dimension<<abo+t 1-! to 1-2 cm larger. C+t o+t the nitrocellose so that it is slightly smaller than the 3hatmann filter papers b+t still covers the entire s+rface of the gel. 5. Float the nitrocell+lose filter on the s+rface of a tray of deioni ed water and allow it to wet from beneath by capillary action. "hen, s+bmerge the filter in the water for five min+tes to displace trapped air b+bbles. !. Remove the plates from the apparat+s. Remove the spacers and pry off one of the plates by inserting a spat+la and twisting the plate +p. "he gel sho+ld now be st+c0 to one of the plates. 8. Caref+lly float the gel off the plate into a "+pperware container containing transfer b+ffer. Let it e4+ilibrate in the transfer b+ffer for 18 min+tes. &. 3hen the gel has e4+ilibrated, constr+ct a @sandwich@ for the transfer as followsA Ba. )lace some transfer b+ffer in a large glass or "+pperware container and place the plastic frame inside so the green side is lying in the b+ffer and the white side is folded o+t. b. )lace a sponge on top of the green side, and then stac0 two sheets of the 3hatmann paper yo+ c+t o+t on top. "hese things sho+ld be s+bmerged in the b+ffer so that they become completely wet. c. )lace the nitrocell+lose filter, labeled appropriately, on top, then caref+lly position the gel on top of this. d. Add two more pieces of 3hatmann filter paper, then a second sponge. "hen press everything down into the b+ffer and s4+ee e o+t any air b+bbles that might be trapped in the sandwich +sing a pipette as a roller. e. Close the sandwich by folding the white side over onto the green and loc0ing it in place. "hen immerse the sandwich in o+r transfer tan0 to which transfer b+ffer has been added. *ince the proteins will migrate toward the positive end, ma0e s+re that the

Citrocell+lose side is closest to the positive electrode. "hat is, the green side of the sandwich sho+ld be facing the red side of the tan0. 9. "ransfer the gel by r+nning at 28 D overnight. $a0e s+re to have the cooling +nit on at ! C so that cold water can circ+late thro+gh the tan0 while the transfer is ta0ing place. 1therwise, if the b+ffer gets hot, b+bbles of air may come o+t of sol+tion and become trapped in the sandwich.

Probing the blot with an antibod

1. Remove the blot from the transfer +nit and bloc0 by placing in 8( mil0 blotting sol+tion for 1 ho+r with sha0ing. 2. 7nc+bate in primary antibody on o+r roc0er for one ho+r. "ypically, the primary antibody is dil+ted 1A1 or so if it is from the s+pernatant of a hybridoma cell line all the way +p to 1A 1''' or more, if say it is p+rified from ascites fl+id. 3e have been dil+ting primary in 8( mil0 blotting sol+tion. *mall vol+mes of 8 mL or less of dil+ted primary antibody can be added to a blot as large as 28' cm2 if the plastic heat sealable bags we have are +sed. Eo+ can c+t and seal the bag so the area aro+nd yo+r blot is minimi ed. Add the primary, smooth o+t b+bbles with a pipette, and seal. 5. 3ash the blot three times with wash b+ffer, 8<1' min+tes each time. !. 7nc+bate the blot in secondary antibody for one ho+r on a roc0er. $ost of o+r primary antibodies are from mice, so the goat antimo+se antibody from the #CL 0it can be +sed. "he blot can be inc+bated in 1' mL of wash b+ffer that is 2( ?*A and to which secondary antibody has been added to a 1A8''' dil+tion. 8. 3ash the blot fo+r times with wash b+ffer, 8<1' min+tes each time. ?etween washes, rinse briefly with F21. &. "oward the end of the washes, prepare for e/posing the blot to film. "a0e an old :@ > 1'@ film and place *aran wrap aro+nd one side. "hen, ta0e a second piece of *aran wrap and place it aro+nd the same side s+ch that there is an opening that the blot can be slid into.

9. 3hen the blot is finished washing, blot the e/cess wash onto a 3hatmann filter. )lace the blot onto a piece of *aran wrap and add the chemil+menescence reagents. As little as 1 mL of each of the two reagents can be mi/ed in a t+be and added to a 28' cm2 blot. Fold the *arans wrap over the blot and ma0e s+re that the small vol+me of reagents trapped in the space in between contacts the entire s+rface of the blot. Leave the reagents on for abo+t one min+te. :. ?lot off the e/cess, shove the blot into the case yo+ constr+cted, and e/pose to film. A one min+te e/pos+re can be tried first as a test e/pos+re. "hen other e/pos+re times can be +sed depending on the intensity yo+ see with this.