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| - | ==Pouring Tris-Tricine Acrylamide Gels== | + | ==Lab Protocols== |
| - | 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)
| + | [[Endy:Tris-Tricine Acrylamide Gels]]<br> |
| - | *5 uL TEMED
| + | [[Sauer:bis-Tris SDS-PAGE, the very best]]<br> |
| - | ==Stacking Gel (4%)==
| + | [[Keating:Experimental Protocols:SDS-PAGE]]<br> |
| - | *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]] |
| - | Add APS and TEMED immediately before pouring.
| + | [[Category:Protein]] |
| - | | + | [[Category:In vitro]] |
| - | 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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