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

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 $189 for the 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!