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SDS-PAGE
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SDS-PAGE Materials: • 6X Sample Buffer (store aliquots at -20 deg C): 6X Composition (for 10 mls) 60% Glycerol 6.0 ml 100% Glycerol 300 mM Tris (pH 6.8) 3.0 ml 1 M Tris 6.8 12 mM EDTA 240 ul 0.5 M EDTA 12% SDS 1.2 g SDS 864 mM 2-mercaptoethanol 600 ul 2-mercaptoethanol 0.05% bromophenol blue "pinch" bromophenol blue • for polyacrylamide gel: 1 M Tris-Cl (pH 8.8) 10X Electrophoresis Buffer 1 M Tris-Cl (pH 6.8) 30 g Tris 20% SDS 145 g Glycine 40% Acrylamide (37.5:1 acrylamide:bis-acrylamide) 10 g SDS 10% Ammonium persulfate (store aliquots @ -20) bring to 1 L with H2O TEMED 2% Agarose 0.01% SDS • for staining/destaining gel: Coomassie Staining Solution Destaining Solution 0.1% Coomassie brilliant blue R-250 50% Methanol 50% Methanol 10% Acetic Acid 10% Acetic Acid Gel Equilibration Buffer (dissolve Coomassie in Methanol before adding water and acetic acid - will require stirring 1-2 hours) 20% Methanol 10% Glycerol Procedure: 1. Pour Polyacrylamide Gel o assemble gel sandwich wash well and rinse (final w/ 95% EtOH) plates, spacers, and combs lay back plate (cut out plate) on flat surface place spacers along bottom and sides of back plate place top plate on top of spacers offset from bottom edge by 1-2 mm clamp together with 1" binder clips seal sandwich along bottom and sides with molten 2% agarose o pour separating gel (5-15%) determine percent gel needed and combine components in 15 ml conical tube Separating Gel Percentage (vol in mls) final stock solution 5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15% conc. 1 M Tris 8.8 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 375 mM 20% SDS 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.1 % 40% Acrylamide 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 5-15 % H2O 4.95 4.70 4.45 4.20 3.95 3.70 3.45 3.20 2.95 2.70 2.45 o add 100 ul 10% APS and 6 ul TEMED (increase to 10ul TEMED if gel percent is less than 8%) o mix well by inverting tube o pour solution into gel sandwich using a Pasteur pippette (run it along a side spacer) until desired level o leave enough room for a stacking gel of 1/2 well depth below the level where the comb teeth reach o gently overlay with 0.01% SDS to create a barrier between the gel and the air o allow gel to polymerize 30-60 minutes at room temp or until interface appears o pour stacking gel (3.75%) pour off aqueous layer from separating gel and rinse with ddH2O combine components for stacking gel: 2.329 mls H2O 0.375 mls Tris 6.8 281 ul 40% Acrylamide mix 15 ul 20% SDS 30 ul 10% APS 5 ul TEMED pour stacking solution on top of separating gel insert comb into stacking gel taking care to avoid forming bubbles on the ends of the teeth allow gel to polymerize 30-60 minutes o clamp gel onto electrophoresis tank carefully remove binding clips and the comb from gel remove bottom spacer (leave the side spacers) place gel against electrophoresis tank with the cut out plate facing the tank and clamp into place with large binding clips fill upper and lower reservoirs with 1X Electrophoresis buffer straighten wills out with tip of Hamilton syringe rinse out wells and bottom of gel with buffer using syringe with bent needle for bottom edge 2. Prepare samples o thaw protein samples rapidly in room temp water bath o add 1/5 vol 6X Sample Buffer to protein samples o boil samples for 5-10 minutes o spin down samples in microfuge for 1 minutes 3. Separate protein samples by PAGE o load samples into wells using Hamilton syringe o fill empty wells with protein extract buffer + Sample dye o attach electrodes so that proteins will move towards the anode (+ or red lead) o run gel at 100-200 V until dye front reaches the bottom of the gel 4. Stain gel to visualize protein bands o disconnect electrodes, dump out electrophoresis buffer, and remove gel sandwich from tank o remove side spacers and carefully pry apart the plates so that the gel remains on one plate o pour staining solution into pyrex dish o invert plate with gel into the staining solution and gently allow gel to "float" off of plate into solution o cover with plastic wrap and gently agitate gel for 60-120 minutes (longer will increase sensitivity but requires more destaining) o remove staining solution (can be reused many times) and rinse gel with ddH2O to remove excess stain o add destaining solution and agitate until satisfied o change destaining solution and agitate until proper level of destaining is achieved (can destain overnight if needed) 5. Preserve gel o remove destaining solution o agitate gel in gel equilibration buffer 15-30 minutes o soak two sheets of cellophane in ddH2O o place one sheet of cellophane on gel drying rack o place gel on top of cellophane, taking care to avoid trapping air bubbles o place second sheet of cellophane on top of gel and remove any trapped air bubbles o place upper rack on top of cellophane-gel sandwich and clamp together o allow to air dry overnight Notes & Miscellaneous • Coomassie staining sensitivity is approximately 100 ng protein • unpolymerized acrylamide is a neurotoxin - wear gloves when handling o once polymerized, gels can be disposed off in the regular trash • keep excess solution to monitor polymerization process • to facillitate visualization of wells in the stacking gel, add bromophenol blue to stacking solution • add KimWipes to destaining solution to soak up dye as it diffuses out of gel (if left in too long, i.e. overnight, can also cause destaining of proteins themselves) • glycerol in equilibration buffer helps prevent cracking during drying of high percentage gels • MW determination: o Rf value = [distance of protein migration] / [distance of tracking dye migration] o plot log(MW) vs Rf for size standards and interpolate unknown protein from its Rf value • offsetting top plate of sandwich allows easy rinsing of gel using a bent 20G needle • insert comb into stacking gel at an angle to help avoid trapping air bubbles at end of comb teeth • "smiling" and "frowning" of gels largely due to unequal heat distribution / salt concentration across gel o run gels slower or with cooling in the cold room o fill empty wells with extract buffer |
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2楼2008-06-26 08:11:43
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