Lysis buffers
These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.RIPA buffer (radioimmunoprecipitation assay buffer)RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent and is particularly useful for nuclear membrane disruption for nuclear extracts. A RIPA buffer gives low background but can denature kinases. It can also disrupt protein-protein interactions and may, therefore, be problematic for immunoprecipitations and pull-down assays.50 mM Tris-HCl, pH 8.0150 mM NaCl1% IGEPAL CA-6300.5% sodium deoxycholate0.1% SDSProtease inhibitorsThe 10% sodium deoxycholate stock solution (5 g into 50 mL) must be protected from light.
NP-40 buffer 150 mM NaCl1.0% NP-40 (possible to substitute with 0.1% Triton X-100)50 mM Tris-HCl, pH 8.0Protease inhibitorsTris-HCl
20mM Tris-HClProtease inhibitorsCytoskeletal bound proteins extract buffer10 mM Tris, pH 7.4100 mM NaCl1 mM EDTA1 mM EGTA1 mM NaF20 mM Na4P2O72 mM Na3VO41% Triton X-10010% glycerol0.1% SDS0.5% deoxycholateSoluble protein buffer20 mM Tris-HCl, pH 7.51 mM EGTA (Ca2+ chelator)
Loading, running, transfer, and blocking buffersLoading buffer/Laemmli 2X buffer
4% SDS10% 2-mercaptoethanol20% glycerol0.004% bromophenol blue0.125 M Tris-HClCheck the pH and adjust it to 6.8Running buffer (Tris-Glycine/SDS)
25 mM Tris base190 mM glycine 0.1% SDSCheck the pH and adjust to 8.3Transfer buffer (wet)
25 mM Tris base190 mM glycine 20% methanolCheck the pH and adjust to 8.3For proteins >80 kDa, we recommend including SDS at a final concentration of 0.1%.Transfer buffer (semi-dry)
48 mM Tris39 mM glycine20% methanol0.04% SDSBlocking buffer
3–5% milk or BSA (bovine serum albumin)Add to the TBST buffer. Mix well and filter. Failure to filter can lead to spotting, where tiny dark grains will contaminate the blot during color development.Sodium orthovanadate preparationAll procedures must be carried out under the fume hood.
Prepare a 100 mM sodium orthovanadate solution with double distilled waterSet pH to 9.0 with HClBoil until colorlessCool to room temperatureSet pH to 9.0 againBoil again until colorlessRepeat this cycle until the solution remains at pH 9.0 after boiling and coolingBring up to the initial volume with waterStore in aliquots at -20°CDiscard if the samples turn yellowAvoid large changes in volume during boiling; put a loose lid on the container to protect from evaporation.
TBS 10x (concentrated Tris-buffered saline)For 1 L:24 g Tris base (formula weight: 121.1 g)88 g NaCl (formula weight: 58.4 g)Dissolve in 900 mL distilled waterpH to 7.6 with 12 N HClAdd distilled water to a final volume of 1 L
For a 1x solution, mix 1 part of the 10x solution with 9 parts distilled water and adjust pH to 7.6 again. The final molar concentrations of the 1x solution are 20 mM Tris and 150 mM NaCl.
An alternative recipe for Tris buffer combines Tris base and Tris-HCl. This avoids the large volume of potentially hazardous hydrochloric acid that is needed to neutralize a solution of Tris base alone.
TBS 10x alternative recipe (concentrated Tris-buffered saline)For 1 L:24 g Tris-HCl (formula weight: 157.6 g)5.6 g Tris base (formula weight: 121.1 g)88 g NaCl (formula weight: 58.4 g)Dissolve in 900 mL distilled water
The pH of the solution should be about 7.6 at room temperature. If too basic, adjust to pH 7.6 with concentrated HCl, and if too acidic, adjust with concentrated NaOH.Add distilled water to a final volume of 1 L.For a 1x solution, mix 1 part 10x with 9 parts distilled water and pH to 7.6 again.The final molar concentrations of the 1x solution are 20 mM Tris and 150 mM NaCl.TBST (Tris-buffered saline, 0.1% Tween 20)For 1 L:100 mL of TBS 10x900 mL distilled water1 mL Tween 20
Medium stripping buffer15 g glycine1 g SDS10 mL Tween 20
Adjust the volume to 800 mL with ultra pure water.Adjust pH to 2.2.Bring volume up to 1 L with distilled water.Harsh stripping bufferThis needs to be done under a fume hood.
For 100 mL:20 mL SDS 10%12.5 mL Tris HCl, pH 6.8, 0.5 M67.5 mL distilled waterAdd 0.8 mL β-mercaptoethanol under the fume hood
Nuclear fractionation protocol reagents buffer A10 mM HEPES1.5 mM MgCl210 mM KCl0.5 DTT0.05% NP-40 (or 0.05% Igepal or Tergitol) pH 7.9
To prepare 250 mL stock of buffer A:HEPES: 1 M = 238.3 g/L, therefore 10 mM = 0.59 g/250 mLMgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mLKCl: 1 M = 74.5 g/L, therefore 10 mM = 0.187 g/250 mLDTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mLNP-40: 0.05%
Nuclear fractionation protocol reagents buffer B5 mM HEPES1.5 mM MgCl20.2 mM EDTA0.5 mM DTT26% glycerol (v/v) pH 7.9
To prepare 250 mL stock of buffer B:HEPES: 1 M = 238.3 g/L, therefore 5 mM = 0.295 g/250 mLMgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mLEDTA: 1 M = 372.2 g/L, therefore 0.2 mM = 0.0186 g/250 mLDTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mL26% glycerol (v/v) = 65 mL
TBS 0.025% Triton X-100For 1 L:250 µL Triton X-1001 L TBS pH 7.6–7.8
1.6% H2O2 (hydrogen peroxide) in TBSFor 400 mL:6.4 mL H2O2 (GPR = 30% w/w)393.6 mL TBS pH 7.6–7.8
Primary antibody made up in TBS with 1% BSAExample is of primary antibody used at a dilution of 1:10.
For 1 mL:100 µL primary antibody10 mg BSA900 µL TBS pH 7.6–7.8
ABC (avidin-biotin complex) in TBSExample is of ABC, each part used at a dilution of 1:100.
For 1 mL:10 µL Streptavidin10 µL HRP (or AP)-biotin980 µL TBS pH 7.6–7.8
Bicarbonate/carbonate coating buffer (100 mM)3.03 g Na2CO36.0 g NaHCO3 (1 L distilled water) pH 9.6PBS: 1.16 g Na2HPO40.1 g KCl0.1 g K3PO44 g NaCl (500 mL distilled water) pH 7.4
Protocols are provided by Abcam “AS-IS” based on experimentation in Abcam’s labs using Abcam’s reagents and products; your results from using protocols outside of these conditions may vary.
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