Acrylamide gels

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Current revision (17:10, 10 August 2006) (view source)
m (Acrylamide Gels moved to Acrylamide gels: capitalization convention)
 
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==Pouring Tris-Tricine Acrylamide Gels==
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==Lab Protocols==
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Protocol by [[Jennifer Braff|jcb]]
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==Running Gel (10%)==
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*4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)
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*5 mL Tris-Cl/SDS pH 8.45
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*1.15 mL MilliQ water
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*3.95 mL 40% glycerol
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*25 uL 10% APS (made fresh)
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[[Endy:Tris-Tricine Acrylamide Gels]]<br>
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*5 uL TEMED
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[[Sauer:bis-Tris SDS-PAGE, the very best]]<br>
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==Stacking Gel (4%)==
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[[Keating:Experimental Protocols:SDS-PAGE]]<br>
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*0.81 mL 30% acrylamide/0.8% bisacrylamide
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*1.55 mL Tris-Cl/SDS pH 8.45
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*3.89 mL MilliQ water
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*40 uL saturated bromophenol blue (optional)
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*37.5 uL 10% APS (0.1g ammonium persulfate/mL H20)
 
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*17.5 uL TEMED
 
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[[Category:Protocol]]
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Add APS and TEMED immediately before pouring. 
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[[Category:Protein]]
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[[Category:In vitro]]
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Pour running (separating) gel (use 20G 1.5 syringe) to 1 cm below bottom of comb (mark short plate) and overlay with isopropanol.  Set for 1 hr, rinse with water (buffer might be better), and pour stacking gel.  Insert combs, being careful to avoid bubbles.  Let set, wrap in damp paper towels, and store 4 C for up to one week.
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15 well, .75 mm gels hold about 15 uL sample per lane.  To run gels (MiniProtean III), put 200 mL Anode Buffer in lower/outer chamber and 125 mL Cathode Buffer in inner/upper chamber.  Remove combs after adding running buffers.  Use a syringe or  x-long gel-loading pipette tip to flush out each well gently with Cathode Buffer to remove any unpolymerized acrylamide.
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Run at about 80 V for a couple of hours.
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== Tris-Tricine Cathode Buffer==
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*12.22 g Tris (0.1 M)
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*17.92 g Tricine (0.1 M)
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*1 g SDS (0.1%)
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*to 1 L with MilliQ H2O (no need to pH)
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== Tris-Tricine Anode Buffer (5L)==
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*121 g Tris (0.2 M)
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*500 mL MilliQ H2O
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*pH to 8.9 with 6N HCl (about 30 mL)
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*to 5 L with MilliQ
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==Tris-Cl/SDS pH 8.45==
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*182 g Tris base in 300 mL MilliQ H2O (Tris is 121.14 g/mol) (hard to dissolve)
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*to pH 8.45 with 3N Hcl
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*MilliQ H2O to 500 mL
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*Filter 0.45 um
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*Add 1.5 g SDS
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Current revision

Lab Protocols

Endy:Tris-Tricine Acrylamide Gels
Sauer:bis-Tris SDS-PAGE, the very best
Keating:Experimental Protocols:SDS-PAGE

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