postheadericon Protocols

Buffers

These buffers are tested and used in my routine laboratory work with success.

Cell Lysis Buffer (CLB)

Reagent Weight/Volume Final concentration
  1 M HEPES (pH 7.4) 2.5 ml 50 mM
  Sodium Chloride (NaCl) 440 mg 150 mM
  10% Triton X-100 5 ml 1%
  50 mM EGTA 1 ml 1 mM
  Glycerol 5 ml 10%
  ddH2O up to 50 ml
Total volume 50 ml
Remarks Store at +4°C

Animal Tissue Homogenization & Lysis Buffer (Modified RIPA)

Reagent Weight/Volume Final concentration
  1 M HEPES (pH 7.4) 2.5 ml 50 mM
  Sodium Chloride (NaCl) 440 mg 150 mM
  10% Triton X-100 5 ml 1%
  50 mM EGTA 1 ml 1 mM
  Glycerol 5 ml 10%
  10% Sodium Deaoxycholate 2.5 ml 0.5%
  10% SDS 0.5 ml 0.1%
  n-Octyl-beta-D-Glucoside 1 g ~ 70 mM
  ddH2O up to 50 ml
Total volume 50 ml
Remarks Store at +4°C Glycerol can be substituted with 250 mM sucrose n-Octyl-beta-D-Glucoside (optional) produces bubbles

Cell Permeabilization Buffer (CPB)

Reagent Weight/Volume
  1 M HEPES (pH 7.4) 2.5 ml
  Sodium Chloride (NaCl) 440 mg
  150 μg/ml Digitonin 7.5 mg
  50 mM EGTA 1 ml
  Glycerol 5 ml
  ddH2O up to 50 ml
Total volume 50 ml
Remarks Store at +4°C

Access my protocol of Cellular Fractionation

Cell Immunoprecipitation buffer (CIB)

Reagent Weight/Volume
  1 M HEPES (pH 7.4) 1 ml
  Sodium Chloride (NaCl) 440 mg
  10% Triton X-100 0.5 ml
  Glycerol 5 ml
  ddH2O up to 50 ml
Total volume 50 ml
Remarks Store at +4°C

TBST Buffer (10x)

Reagent Weight/Volume
  Sodium Chloride (NaCl) 87.66 g
  100% Triton X-100 5 ml
  1 M Tris (pH 8.0) 100 ml
  ddH2O up to 1 L
Total volume 1 L
Remarks Store at RT or +4°C

NuPAGE Laemmli Sample

Reagent Weight/Volume
  4x LDS Sample Buffer 25 μl
  0.5 M Dithiothreitol (DTT) 10 μl
  Cell lysate 65 μl
Total volume 100 μl
Remarks Heat 75°C for 5 min

Antioxidant

Reagent Weight/Volume Final concentration
  Sodium bisulfite 7.5 g/40 ml ddH20 15%
  N,N-Dimethylformamide 5 ml 10%
  ddH2O up to 50 ml
Total volume 50 ml
Remarks Don't shake!
Seal the tube!
Store at +4°C Crystals are OK

10 x Reducing Reagent

Reagent Weight/Volume Final concentration
  DTT 3.85 g 0.5 M
  ddH2O up to 50 ml
Total volume 50 ml
Remarks Store in amber tube! Store at +4°C Prepare fresh every 2 weeks

MOPS-SDS Running buffer (20x)

Reagent Weight/Volume Final concentration
  MOPS 209.26 g 1 M
  Tris 121.14 g 1 M
Adjust pH to 7.7! EDTA 5.845 g 20 mM
  SDS 20 g 2 %
  ddH2O up to 1 L
Total volume 1 L
Remarks Add SDS only after adjusting pH! Store at RT Yellow buffer is OK

NuPAGE Running Buffer (1x)

Reagent Weight/Volume
  NuPAGE MOPS-SDS (20x) 50 ml
  ddH2O 950 ml
  Antioxidant 500 μl
Total volume 1 L
Remarks Store at +4°C

NuPAGE protein Transfer buffer (20x)

Reagent Weight/Volume Final concentration
  Bicine 81.6 g 25 mM
  Bis-Tris (free base) 104.62 g 25 mM
EDTA 5.84 g 1 mM
  Chlorobutanol (optional) 1 mL 0.05 mM
  ddH2O up to 1 L
Total volume 1 L
Remarks pH should be ~ 7.2 Store at +4°C  

NuPAGE Transfer Buffer (1x)

Reagent Weight/Volume
  20x NuPage transfer buffer 50 ml
  ddH2O 850 ml
  Methanol 100 ml
  Antioxidant 1 ml
Total volume 1 L
Remarks Store at +4°C

SDS-PAGE Transfer Buffer

Reagent Weight/Volume
  10x Tris-Glycine 60 ml
  10x Tris-Glycine-SDS 20 ml
  ddH2O 720 ml
  Methanol 200 ml
Total volume 1 L
Remarks Store at +4°C

10x Tris-Glycine

Reagent Weight/Volume Final concentration
  Tris 30.285 g 0.25 M
  Glycine 144.134 g 1.92 M
  ddH2O up to 1 L
Total volume 1 L
Remarks Adjust pH to 8.5 Store at +4°C

10x Tris-Glycine-SDS

Reagent Weight/Volume Final concentration
  Tris 30.285 g 0.25 M
  Glycine 144.134 g 1.92 M
  ddH2O up to 1 L
  SDS 10 g 1%
Total volume 1 L
Remarks Adjust pH to 8.3
Then add SDS Store at +4°C

Inhibitor cocktails

Phosphatase inhibitor cocktail (100x)

Reagent Mr (g/M) Weight/Volume Stock concentration
  Sodium fluoride 42 84 mg 200 mM
  Imidazole 68.1 136.2 mg 200 mM
  Sodium molybedate 205.9 236.785 mg 115 mM
  Sodium orthovanadate 183.9 5 mL (see Remarks) 200 mM
  Sodium tartrate dihydrate 230.1 920.4 mg 400 mM
  Sodium pyrophosphate 416.1 416.1 mg 100 mM
  β-Glycerophosphate 306.1 306.1 mg 100 mM
  ddH2O 5 mL
Total volume 10 ml
Remarks Na3VO4 recipe: Aliquot 1 mL Store at -20°C

1) Prepare a solution of 200 mM sodium orthovanadate in ultrapure dH20 according to protocol by Gordon (1991) PubMed. For a 100 mL solution, add 3.68 g Na3VO4 to 90 mL water and dissolve with stirring. Once dissolved, bring volume to 100 mL.
2) Depending on the pH of the solution, slowly add either 1 M NaOH or 1 M HCl with stirring to adjust pH to 10. Adding HCl will make the solution yellow.
3) Boil solution by heating in a microwave for 5 - 15 sec. After boiling for 5 - 15 sec, the solution will be clear and colorless.
4) Cool on ice until the Na3VO4 solution reaches room temperature.
5) At this point, the pH will be greater than 10. Add a small amount (several drops, with stirring) of 1 M HCl to adjust solution pH to 10.
6) Repeat steps 3-5 a total of 3-5 times. After several cycles of boiling, cooling, and adjusting pH, the solution should reach a point where the pH stablizes at ~10. At this point, adding HCl should result in little, if any, appearance of yellow color in the solution.
7) Aliquot and store activated Na3VO4 at -20°C.

Alamar Blue (AB) assay

It is ridiculous to pay $189 dollars for 25 mL of Alamar Blue solution. It is a rip-off of scientists by greedy biotech companies, who rely on the myth (or maybe on a sad but true reality) that most PIs, postdocs and students are lazy, stinkin' rich and do not want to make reagents on their own. I never buy if i can make it on my own using raw materials. For the money saved I can buy something I can't make (e.g. inhibitors, ligands).

STOCK SOLUTION: Dissolve 1 g resazurin sodium salt in 100 mL sterile PBS. Sterile filter through a 0.22 µm filter. Store at +4°C forever.

1x SOLUTION: Take 150-200 µL of your stock solution and add 50 mL sterile PBS (you may adjust this volume if you want less intense (150 µL), optimal (180 µL) or more intense (200 µL) initial AB color, which would affect fluorescent color development time as well). Completely remove the media by vacuum suction from growing adherent cells (do not touch well surface!!!) and quickly add 1x solution (do not dilute) in a multiwell plate (use multi-channel pipettor and sterile disposable plastic reagent reservoir). Make sure you have at least 3 blank wells without cells for subsequent subtraction of background. Measure fluorescence using 540/35 excitation filter and a 590/20 emission filter or absorbance at 570 nm and 600 nm using the microplate reader of choice.

SAVINGS: 1 g resazurin (MW: 251.17; Cas No: 62758-13-8) from Acros Organics costs $18.32, and when buying 5 g you get even more savings - it costs $50.59, and if you want the ultimate profit - buy 25 g for $174.56 - now this option is a lifetime supply of AB for you, your children and your grandchildren regardless how many plates they will assay in their lifetime (if you are not convinced then do the math: 1 g of Resazurin stock solution provides at least 1000 of 25 ML 1x AB tubes). Do you still want to pay LifeTechnologies $189 for their pony size 25 mL?

Luminol Chemiluminescent detection solution

The only major disadvantage of sensitive luminol-based solutions is their outrageous pricing. The recipe of competitive home-made solution and comparative testing results will come soon! Stay connected!