Protocol: Protein electrophoresis and western blot recipes

1 Protein electrophoresis and western blot recipesStock solutions 1 M Tris-HCl, pH M Tris-HCl, pH 10% SDS bromophenol blue 10X Tris-buffered saline (TBS) 10X phosphate-buffered saline (PBS)Sample preparation buffers RIPA buffer 2X SDS sample buffer (Laemmli buffer) 4X LDS sample bufferElectrophoresis running buffers 10X Tris-glycine SDS running buffer 10X Tris-glycine native running buffer 20X MOPS SDS running buffer 20X MES SDS running buffer 10X tricine SDS running bufferTransfer buffer 25X Tris-glycine transfer buffer 20X Bis-Tris transfer bufferWash buffers Tris-buffered saline with Tween 20 surfactant (TBST) Phosphate-buffered saline with Tween 20 surfactant (PBST)Blocking and stripping buffers 5% nonfat milk 3% BSA Stripping bufferGel casting recipes Invitrogen SureCast reagents Stand-alone reagentsStock solutions Sample preparation buffers Ready-to-use alternatives:Thermo Scientific Pierce 20X TBS Buffer (Cat.)

2 No. 28358)Thermo Scientific Pierce 20X PBS Buffer (Cat. No. 28348)Ready-to-use alternative:Invitrogen Novex Tris-Glycine SDS Sample Buffer (2X) (Cat. No. LC2676)Ready-to-use alternative:Thermo Scientific RIPA Lysis and Extraction Buffer (Cat. No. 89900)1 M Tris-HCl, pH Tr is ba gDeionized water80 mLAdjust to pH with HClDeionized waterto 100 M Tris-HCl, pH Tr is ba gDeionized water60 mLAdjust to pH with HClDeionized waterto 100 mL10X Tris-buffered saline (TBS)Tr is ba se24 gNaCl88 gDeionized water900 mLAdjust to pH with HClDeionized water to 1,000 mL10X phosphate-buffered saline (PBS) NaCl80 gKCl2 gDeionized water900 mL Adjust to pH with NaOHDeionized waterto 1,000 mL RIPA buffer: 25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, SDS (100 mL), pH gNP-401 gSodium deoxycholate1 g10% SDS1 mL1 M Tris-HCl, pH mLDeionized waterto 100 mLThermo Scientific Pierce Protease Inhibitor Tablet (Cat.

3 No. A32965)2 tablets SDS sample buffer (Laemmli buffer): 63 mM Tris-HCl, 10% glycerol, 2% SDS, bromophenol blue, pH Recipe for 2X buffer M Tris-HCl, pH mLGlycerol 2 mL10% (w/v) SDS4 (w/v) bromophenol mLDeionized waterto 10 mL The buffer is stable for 6 months when stored at 4 C. 10% SDS 0 gDeionized waterto 10 mL bromophenol blue Bromophenol blue100 mgDeionized waterto 10 mL Sample preparation buffers electrophoresis running buffers Ready-to-use alternative:Invitrogen NuPAGE LDS Sample Buffer (4X) (Cat. No. NP0007)Ready-to-use alternative: Invitrogen Novex Tris-Glycine SDS Running Buffer (10X) (Cat. No. LC2675)Ready-to-use alternative: Invitrogen Novex Tris-Glycine Native Running Buffer (10X) (Cat. No. LC2672)Ready-to-use alternative: Invitrogen NuPAGE MOPS SDS Running Buffer (20X) (Cat. No. NP0001)LDS sample buffer: 106 mM Tris-HCl, 141 mM Tris base, 2% LDS, 10% glycerol, mM EDTA, mM SERVA Blue G250, mM phenol red, pH Recipe for 4X buffer stock:Tr is gTr is ba gGlycerol4 gSERVA Blue G250 (1% solution) mLPhenol red (1% solution) mLDeionized water to 10 mL The buffer is stable for 6 months when stored at 4 C.

4 Do not use acid or base to adjust pH. Tris-glycine SDS running buffer: 25 mM Tris base, 192 mM glycine, SDS, pH Recipe for 10X buffer stock:Tr is ba se29 gGlycine144 gSDS10 gDeionized waterto 1,000 mLTris-glycine native running buffer: 25 mM Tris base, 192 mM glycine, pH for 10X buffer stock:Tr is ba se29 gGlycine144 gDeionized waterto 1,000 mLMOPS SDS running buffer: 50 mM MOPS, 50 mM Tris base, SDS, 1 mM EDTA, pH Recipe for 20X buffer gTr is ba gSDS10 gDeionized waterto 500 mLElectrophoresis running buffers Transfer buffersReady-to-use alternative: Invitrogen NuPAGE MES SDS Running Buffer (20X) (Cat. No. NP0002)Ready-to-use alternative: Invitrogen Novex Tricine SDS Running Buffer (10X) (Cat. No. LC1675) Ready-to-use alternative: Invitrogen NuPAGE Transfer Buffer (20X) (Cat. No. NP0006)Ready-to-use alternative: Invitrogen Novex Tris-Glycine Transfer Buffer (25X) (Cat.)

5 No. LC3675)MES SDS running buffer: 50 mM MES, 50 mM Tris base, SDS, 1 mM EDTA, pH for 20X buffer stock:MES9 7. 6 gTr is ba gSDS10 gDeionized waterto 500 mLDo not use acid or base to adjust pH. Tricine SDS running buffer: 100 mM Tris base, 100 mM tricine, SDS, pH Recipe for 10X buffer stock:Tr is ba se121 gTricine179 gSDS10 gDeionized waterto 1,000 mLThe buffer is stable for 6 months when stored at room temperature. Do not use acid or base to adjust pH. Bis-Tris transfer buffer: 25 mM bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, pH Recipe for 20X buffer gB is-Tr is (f re e ba se)13 .1 gDeionized waterto 125 mLThe buffer is stable for 6 months when stored at 4 C. Do not use acid or base to adjust pH. Tris-glycine transfer buffer: 12 mM Tris base, 96 mM glycine, pH Recipe for 25X buffer stock:Tr is ba gGlycine90 gDeionized waterto 500 mLWash buffers Blocking and stripping buffers Ready-to-use alternative: Thermo Scientific Pierce 20X PBS Tween 20 Buffer (Cat.

6 No. 28352)Ready-to-use alternative: Thermo Scientific Pierce Blocker BSA (10X) in TBS (Cat. No. 37520)Thermo Scientific Pierce Blocker BSA (10X) in PBS (Cat. No. 37525)Ready-to-use alternative: Thermo Scientific Pierce 20X TBS Tween 20 Buffer (Cat. No. 28360)Ready-to-use alternative: Thermo Scientific Pierce Clear Milk Blocking Buffer 10X (Cat. No. 37587)Ready-to-use alternatives: Thermo Scientific Restore western Blot Stripping Buffer (Cat. No. 21059)Thermo Scientific Restore Fluorescent western Blot Stripping Buffer (Cat. No. 62300)Phosphate-buffered saline with Tween 20 surfactant (PBST) 10X PBS 100 mLTween 20 surfactant1 mLDeionized waterto 1,000 mL3% BSA gTBST or PBSTto 50 mLFilter to remove particulatesTris-buffered saline with Tween 20 surfactant (TBST)10X TBS100 mLTween 20 surfactant1 mLDeionized waterto 1,000 mL5% nonfat milk Nonfat dry gTBST or PBSTto 50 mLFilter to remove particulatesStripping buffer M Tris-HCl, pH mL10% SDS20 mLDeionized water6 7.

7 5 m L Gel casting recipes recipes for SureCast reagentsThe volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast. recipes for stand-alone reagents Stock solutionsPrepare the following stock solutions. All solutions can be stored at room temperature. Polyacrylamide concentrationSeparating gel solution 4%6%8%10%12%14%16%18%20%SureCast Acrylamide (40%) (Cat. No. HC2040) mLSureCast Resolving Buffer (Cat. No. HC2215) mLDistilled water5 .1 m mL3 .1 m mL10% SureCast APS (Cat. No. HC2005)80 L80 L80 L80 L80 L80 L80 L80 L80 LSureCast TEMED* (Cat. No. HC2006)8 L8 L8 L8 L8 L8 L8 L8 L8 L* Add this last and mix well just before the gel is to be the stacking gel solution according to the following table. The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast.

8 Note: Solutions do not require gel solution4% SureCast Acrylamide (40%) (Cat. No. HC2040) mLSureCast Stacking Buffer (Cat. No. HC2112) mLDistilled mL10% SureCast APS (Cat. No. HC2005)30 LSureCast TEMED* (Cat. No. HC2006)3 L* Add this last and mix well just before the gel is to be acrylamide/Bis (29:1) g acrylamide g BisBring to 100 mL with waterStore up to two months in a dark glass bottle Separating gel buffer (1 M Tris-HCl, pH ) Add g Tris to 150 mL water Adjust to pH with HCl Bring to 250 mL with waterStacking gel buffer ( M Tris-HCl, pH ) Add g Tris to 150 mL water Adjust to pH with HClBring to 250 mL with waterCatalyst: ammonium persulfate (APS) (make fresh the day of use) 100 mg ammonium persulfateBring to 2 mL with water10% SDS g SDSB ring to 100 mL with water50% sucrose g sucroseBring to 100 mL with waterSeparating gelThe volumes provided in each column are for approximately 25 mL of separating gel, enough for four mm thick mini gels.

9 Scale volumes proportionally based on the number of gels to be gel The following recipe is for a 4% stacking gel ( mL).Solution 6% gel 8% gel10% gel12% gel14% gel16% gel18% gel20% gel50% mL7. 0 m mLSeparating gel mL10% SDS250 L250 L250 L250 L250 L250 L250 L250 L50% sucrose* mLWater7. 8 m mL750 LTEMED** LAPS**625 L625 L625 L625 L625 L625 L625 L625 L* Optional but recommended because it makes it easy to form a good interface between the separating gel and the overlay. If omitted, increase the amount of water added to make up for the volume of the sucrose solution (increase the water by mL for the above tables).** Add these last and mix well just before the gel is to be 4% 50% mLStacking gel mL10% SDS125 mLTEMED** LAPS** mL** Add these last and mix well just before the gel is to be Research Use Only.

10 Not for use in diagnostic procedures. 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Tween is a trademark of Croda International Plc. C O L118 3 2 0 4 2 0 Find out more at selection and educational support to help youget better western blotsHaving problems with your western blot?Count on our technical support scientists for experienced troubleshooting America: (800) 955-6288, ext. 2-3 | : 00 800 5345 5345 | of North America and Europe: BlotBuilder western blot product selection toolThis interactive tool is designed to help you select the optimal products for your western blotting needs. Simply answer five questions about your Protein of interest and review a set of recommended products along with a personalized western blotting electrophoresis and western blotting education centerGetting publication-quality western blot results can be a challenge.