WIXLIB

Protein electrophoresis and westernblot recipesStock solutions 1 M Tris-HCl, pH 7.6Transfer buffer 25X Tris-glycine transfer buffer 0.5 M Tris-HCl, pH 6.8 20X Bis-Tris transfer buffer 10% SDS 1.0% bromophenol blueWash buffers Tris-buffered saline with Tween 20 surfactant (TBST) 10X Tris-buffered saline (TBS) Phosphate-buffered saline with Tween 20 surfactant (PBST) 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 bufferBlocking and stripping buffers 5% nonfat milk 3% BSA Stripping bufferGel casting recipes Invitrogen SureCast reagents Stand-alone reagents

Stock solutionsSample preparation buffers1 M Tris-HCl, pH 7.6Tris base12.11 gDeionized water80 mLAdjust to pH 7.6 with HClDeionized waterto 100 mL0.5 M Tris-HCl, pH 6.8RIPA buffer: 25 mM Tris-HCl, 150 mM NaCl, 1% NP-40,1% sodium deoxycholate, 0.1% SDS (100 mL), pH 7.6NaCl0.88 gNP-401gSodium deoxycholate1g10% SDS1 mL1 M Tris-HCl, pH 7.62.5 mLTris base6.06 gDeionized waterto 100 mLDeionized water60 mLThermo Scientific Pierce ProteaseInhibitor Tablet (Cat. No. A32965)2 tabletsAdjust to pH 6.8 with HClDeionized waterto 100 mL10% SDSSDS1.00 gDeionized waterto 10 mLReady-to-use alternative:Thermo Scientific RIPA Lysis and ExtractionBuffer (Cat. No. 89900)1.0% bromophenol blueBromophenol blue100 mgDeionized waterto 10 mL10X Tris-buffered saline (TBS)Tris base24 gNaCl88 gDeionized water900 mLSDS sample buffer (Laemmli buffer): 63 mM Tris-HCl,10% glycerol, 2% SDS, 0.0025% bromophenol blue,pH 6.8Recipe for 2X buffer stock:0.5 M Tris-HCl, pH 6.82.5 mLAdjust to pH 7.6 with HClGlycerol2 mLDeionized water10% (w/v) SDS4 mL0.1% (w/v) bromophenol blue0.5 mLDeionized waterto 10 mLto 1,000 mL10X phosphate-buffered saline (PBS)NaCl80 gKCl2gNa 2HPO414.4 gNaH2PO42.4 gDeionized water900 mLThe buffer is stable for 6 months when stored at 4 C.Ready-to-use alternative:Adjust to pH 7.0 with NaOHDeionized waterto 1,000 mLReady-to-use alternatives:Thermo Scientific Pierce 20X TBS Buffer(Cat. No. 28358)Thermo Scientific Pierce 20X PBS Buffer(Cat. No. 28348)Invitrogen Novex Tris-Glycine SDS SampleBuffer (2X) (Cat. No. LC2676)

Sample preparation buffersElectrophoresis running buffersLDS sample buffer: 106 mM Tris-HCl, 141 mM Tris base,2% LDS, 10% glycerol, 0.51 mM EDTA,0.22 mM SERVA Blue G250, 0.175 mM phenol red, pH 8.5Recipe for 4X buffer stock:Tris-glycine SDS running buffer: 25 mM Tris base,192 mM glycine, 0.1% SDS, pH 8.3Recipe for 10X buffer stock:Tris base29 gTris HCl0.666 gGlycine144 gTris base0.682 gSDS10 gLDS0.800 gDeionized waterto 1,000 mLEDTA0.006 gGlycerol4gSERVA Blue G250 (1% solution)0.75 mLPhenol red (1% solution)0.25 mLDeionized waterto 10 mLThe buffer is stable for 6 months when stored at 4 C. Do not use acid or base to adjust pH.Ready-to-use alternative:Invitrogen NuPAGE LDS Sample Buffer (4X)(Cat. No. NP0007)Ready-to-use alternative:Invitrogen Novex Tris-Glycine SDS RunningBuffer (10X) (Cat. No. LC2675)Tris-glycine native running buffer: 25 mM Tris base,192 mM glycine, pH 8.3Recipe for 10X buffer stock:Tris base29 gGlycine144 gDeionized waterto 1,000 mLReady-to-use alternative:Invitrogen Novex Tris-Glycine Native RunningBuffer (10X) (Cat. No. LC2672)MOPS SDS running buffer: 50 mM MOPS,50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.7Recipe for 20X buffer stock:MOPS104.6 gTris base60.6 gSDS10 gEDTA3.0 gDeionized waterto 500 mLReady-to-use alternative:Invitrogen NuPAGE MOPS SDS RunningBuffer (20X) (Cat. No. NP0001)

Electrophoresis running buffersTransfer buffersMES SDS running buffer: 50 mM MES,50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.3Recipe for 20X buffer stock:Tris-glycine transfer buffer: 12 mM Tris base,96 mM glycine, pH 8.3Recipe for 25X buffer stock:MES97.6 gTris base18.2 gTris base60.6 gGlycine90 gSDS10 gDeionized waterto 500 mLEDTA3.0 gDeionized waterto 500 mLDo not use acid or base to adjust pH.Ready-to-use alternative:Ready-to-use alternative:Invitrogen NuPAGE MES SDS Running Buffer(20X) (Cat. No. NP0002)Tricine SDS running buffer: 100 mM Tris base,100 mM tricine, 0.1% SDS, pH 8.3Recipe for 10X buffer stock:Invitrogen Novex Tris-Glycine TransferBuffer (25X) (Cat. No. LC3675)Bis-Tris transfer buffer: 25 mM bicine,25 mM Bis-Tris (free base), 1 mM EDTA, pH 7.2Recipe for 20X buffer stock:Bicine10.2 gBis-Tris (free base)13.1 gEDTA0.75 gTris base121 gDeionized waterto 125 mLTricine179 gThe buffer is stable for 6 months when stored at 4 C. Do not use acid or base to adjust pH.SDS10 gDeionized waterto 1,000 mLThe buffer is stable for 6 months when stored at room temperature. Do not use acid or base toadjust pH.Ready-to-use alternative:Invitrogen Novex Tricine SDS RunningBuffer (10X) (Cat. No. LC1675)Ready-to-use alternative:Invitrogen NuPAGE Transfer Buffer (20X)(Cat. No. NP0006)

Wash buffersBlocking and stripping buffersTris-buffered saline with Tween 20 surfactant (TBST)5% nonfat milk10X TBS100 mLNonfat dry milk2.5 gTween 20 surfactant1 mLTBST or PBSTto 50 mLDeionized waterto 1,000 mLFilter to remove particulatesReady-to-use alternative:Ready-to-use alternative:Thermo Scientific Pierce 20X TBS Tween 20Buffer (Cat. No. 28360)Phosphate-buffered saline with Tween 20surfactant (PBST)Thermo Scientific Pierce Clear Milk BlockingBuffer 10X (Cat. No. 37587)3% BSABSA1.5 gto 50 mL10X PBS100 mLTBST or PBSTTween 20 surfactant1 mLFilter to remove particulatesDeionized waterto 1,000 mLReady-to-use alternative:Ready-to-use alternative:Thermo Scientific Pierce 20X PBS Tween 20Buffer (Cat. No. 28352)Thermo Scientific Pierce Blocker BSA (10X) in TBS(Cat. No. 37520)Thermo Scientific Pierce Blocker BSA (10X) in PBS(Cat. No. 37525)Stripping buffer0.5 M Tris-HCl, pH 6.812.5 mL10% SDS20 mL2-mercaptoethanol0.8 mLDeionized water67.5 mLReady-to-use alternatives:Thermo Scientific Restore Western BlotStripping Buffer (Cat. No. 21059)Thermo Scientific Restore Fluorescent WesternBlot Stripping Buffer (Cat. No. 62300)

Gel casting recipesRecipes 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.Polyacrylamide concentrationSeparating gel solution4%6%8%10%12%14%16%18%20%SureCast Acrylamide (40%) (Cat. No. HC2040)0.8 mL1.2 mL1.6 mL2.0 mL2.4 mL2.8 mL3.2 mL3.6 mL4.0 mLSureCast Resolving Buffer (Cat. No. HC2215)2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mLDistilled water5.1 mL4.7 mL4.3 mL3.9 mL3.5 mL3.1 mL2.7 mL2.3 mL1.9 mL10% SureCast APS (Cat. No. HC2005)80 µL80 µL80 µL80 µL80 µL80 µL80 µL80 µL80 µLSureCast TEMED* (Cat. No. HC2006)8 µL8 µL8 µL8 µL8 µL8 µL8 µL8 µL8 µL* Add this last and mix well just before the gel is to be poured.Prepare the stacking gel solution according to the followingtable. The volumes provided in the table are for a single gel.Scale volumes proportionally based on the number of gelsto be cast. Note: Solutions do not require degassing.Stacking gel solution4%SureCast Acrylamide (40%) (Cat. No. HC2040)0.30 mLSureCast Stacking Buffer (Cat. No. HC2112)0.75 mLDistilled water1.92 mL10% SureCast APS (Cat. No. HC2005)30 µLSureCast TEMED* (Cat. No. HC2006)3 µL* Add this last and mix well just before the gel is to be poured.Recipes for stand-alone reagentsStock solutionsPrepare the following stock solutions. All solutions can be stored at room temperature.50% acrylamide/Bis (29:1) 48.3 g acrylamide 1.7 g BisBring to 100 mL with waterStore up to two months in adark glass bottleSeparating gel buffer (1 M TrisHCl, pH 8.8) Add 30.3 g Tris to 150 mL water Adjust to pH 8.8 with HClBring to 250 mL with water10% SDS 10.0 g SDSBring to 100 mL with water50% sucrose 50.0 g sucroseBring to 100 mL with waterStacking gel buffer(0.375 M Tris-HCl, pH 6.8) Add 11.4 g Tris to 150 mLwater Adjust to pH 6.8 with HClBring to 250 mL with waterCatalyst: ammoniumpersulfate (APS) (makefresh the day of use) 100 mg ammoniumpersulfateBring to 2 mL with water

Separating gelThe volumes provided in each column are for approximately 25 mL of separating gel, enough for four 1.0 mm thick minigels. Scale volumes proportionally based on the number of gels to be cast.Solution6% gel8% gel10% gel12% gel14% gel16% gel18% gel20% gel50% acrylamide/Bis3.0 mL4.0 mL5.0 mL6.0 mL7.0 mL8.0 mL9.0 mL10.0 mLSeparating gel buffer9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL10% SDS250 µL250 µL250 µL250 µL250 µL250 µL250 µL250 µL50% sucrose*4.0 mL4.0 mL4.0 mL4.0 mL4.0 mL4.0 mL4.0 mL4.0 mLWater7.8 mL6.8 mL5.8 mL4.8 mL3.7 mL2.7 mL1.7 mL750 µLTEMED**6.25 µL6.25 µL6.25 µL6.25 µL6.25 µL6.25 µL6.25 µL6.25 µLAPS**625 µL625 µL625 µL625 µL625 µL625 µL625 µL625 µL* 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 thesucrose solution (increase the water by 4.0 mL for the above tables).** Add these last and mix well just before the gel is to be poured.Stacking gelThe following recipe is for a 4% stacking gel (12.5 mL).Solution4%50% acrylamide/Bis1.0 mLStacking gel buffer4.2 mL10% SDS125 µLWater6.3 mLTEMED**5.0 µLAPS**1.0 mL** Add these last and mix well just before the gel is to be poured.

Product selection and educational support to help youget better western blotsInvitrogen BlotBuilder westernblot product selection toolProtein electrophoresis and westernblotting education centerThis interactive tool is designed to help you select theoptimal products for your western blotting needs. Simplyanswer five questions about your protein of interest andreview a set of recommended products along with apersonalized western blotting protocol.Getting publication-quality western blot results can bea challenge. Access resources to learn about proteingel electrophoresis and western blotting methods, fromwebinars and application notes to quick tips. Whether youare new to western blotting or an experienced researcherwanting to confirm your knowledge, consider this center tohelp you get better western results and succeed sooner.Having problems with your western blot?Count on our technical support scientists for experienced troubleshooting advice.North America:(800) 955-6288, ext. 2-3techsupport@thermofisher.com thermofisher.com/contactusEurope:00 800 5345 5345eurotech@thermofisher.com thermofisher.com/contactusOutside of North America and Europe:thermofisher.com/contactusFind out more at thermofisher.com/westernFor Research Use Only. Not for use in diagnostic procedures. 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarksare the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Tween is a trademark of Croda International Plc.COL11832 0420

Bis-Tris transfer buffer: 25 mM bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, pH 7.2 Recipe for 20X buffer stock: Bicine 10.2 g Bis-Tris (free base) 13.1 g EDTA 0.75 g Deionized water to 125 mL The 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,