All tags Western blot protocols Buffer and stock solutions for western blot

Buffer and stock solutions for western blot


​RIPA 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 8.0
150 mM NaCl
​1% NP-40
​0.5% sodium deoxycholate
​0.1% SDS

​The 10% sodium deoxycholate stock solution (5 g into 50 mL) must be protected from light.



NP-40 buffer

20 mM Tris HCl, pH 8.0
137 mM NaCl
10% glycerol
1% NP-40
2 mM EDTA



Cytoskeletal bound proteins extract buffer

10 mM Tris, pH 7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1 mM NaF
​20 mM Na4P2O7
2 mM Na3VO4
1% Triton X-100
10% glycerol
0.1% SDS
0.5% deoxycholate



Soluble protein buffer

20 mM Tris-HCl, pH 7.5
1 mM EGTA (Ca2+​ chelator)



Sodium orthovanadate preparation

All procedures must be carried out under the fume hood.

  1. Prepare a 100 mM sodium orthovanadate solution with double distilled water
  2. Set pH to 9.0 with HCl
  3. Boil until colorless
  4. Cool to room temperature
  5. Set pH to 9.0 again
  6. Boil again until colorless
  7. Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling
  8. Bring up to the initial volume with water
  9. Store in aliquots at -20°C
  10. Discard if the samples turn yellow


Avoid 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 water
pH to 7.6 with 12 N HCl
Add 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

  1. 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.
  2. Add distilled water to a final volume of 1 L.
  3. For a 1x solution, mix 1 part 10x with 9 parts distilled water and pH to 7.6 again.
  4. 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 10x
900 mL distilled water
1 mL Tween 20



Medium stripping buffer

15 g glycine
1 g SDS
10 mL Tween 20

  1. Adjust the volume to 800 mL with ultra pure water.
  2. Adjust pH to 2.2.
  3. Bring volume up to 1 L with distilled water.



Harsh stripping buffer

This 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 M
67.5 mL distilled water
​Add 0.8 mL β-mercaptoethanol under the fume hood



Nuclear fractionation protocol reagents buffer A

10 mM HEPES
​1.5 mM MgCl2
10 mM KCl
0.5 DTT
​0.05% 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 mL
MgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mL
KCl: 1 M = 74.5 g/L, therefore 10 mM = 0.187 g/250 mL
DTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mL
NP-40: 0.05%



Nuclear fractionation protocol reagents buffer B

5 mM HEPES
1.5 mM MgCl2
​0.2 mM EDTA
0.5 mM DTT
26% 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 mL
MgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mL
EDTA: 1 M = 372.2 g/L, therefore 0.2 mM = 0.0186 g/250 mL
DTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mL
​26% glycerol (v/v) = 65 mL



TBS 0.025% Triton X-100

For 1 L:
250 µL Triton X-100
1 L TBS pH 7.6–7.8



1.6% H2O2 (hydrogen peroxide) in TBS

For 400 mL:
6.4 mL H2O(GPR = 30% w/w)
393.6 mL TBS pH 7.6–7.8



Primary antibody made up in TBS with 1% BSA

Example is of primary antibody used at a dilution of 1:10.

For 1 mL:
​100 µL primary antibody
10 mg BSA
900 µL TBS pH 7.6–7.8



Secondary biotinylated antibody made up in TBS with 1% BSA

Example is of secondary biotinylated antibody used at a dilution of 1:200.

For 1 mL:
5 µL secondary biotinylated antibody
995 µL TBS pH 7.6–7.8



ABC (avidin-biotin complex) in TBS

Example is of ABC, each part used at a dilution of 1:100.

For 1 mL:
10 µL Streptavidin
​10 µL HRP (or AP)-biotin
980 µL TBS pH 7.6–7.8



Bicarbonate/carbonate coating buffer (100 mM)

3.03 g Na2CO3
6.0 g NaHCO​3 (1 L distilled water) pH 9.6
​PBS: 1.16 g Na2HPO4
0.1 g KCl
​0.1 g K3PO4
​4 g NaCl (500 mL distilled water) pH 7.4


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