Western blot | Buffer preparation

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

Buffer preparation

1. Lysis buffer-cell/tissue

Step 1: Preparation of 1 M of Tris-HCl (pH 7.4) stock solution

• Dissolve 121.1 g Tris-base in 800 ml distilled water, adjust the pH to 7.4 using HCl solution, and make up the volume to 1000 mL using distilled water.
• Autoclave to sterilize.

Step 2: Preparation of 100 mL 1M NaCl

• Dissolve 5.844 g NaCl in 80 mL of H2O. and make up the volume to 100 mL using distilled water.

Step 3: Preparation of Sodium Deoxycholate, NaDc (10％, 50ml)

• Dissolve 5 g Sodium Deoxycholate in 25 mL D.W. and make up the volume to 50 mL. Filtration and store.

Step 4: Preparation of 200 mM Sodium orthovanadate, Na3VO4 (200 mM, 50 mL)

• Dissolve Sodium orthovanadate 1.84 gram in 45 mL D.W.

• pH 10 (yellow) and boiling (until colorless), 10 min
• cool to R / T
• pH 10, Boiling for 10 min
• cool to R / T and pH 10
• finally, make up the volume to 50 ml,
• aliquot by 1ml and store at -20 ℃

Step 5: Preparation of Sodium Fluoride, NaF ( 100mM, 50ml)

• Dissolve 0.21 gram  Sodium Fluoride (MW: 41.99) 0.21 gram in 25 mL of D.W.
• finally, make up the volume to 50 ml,
• Filtration, and
• Storage in room temperature.

Step 5: Preparation of Protease inhibitor cocktail, PIC, (×7)

• Dissolve Protease inhibitor cocktail tablet 1 in 1.5 ml D.W.
• Vortexing, aliquot stored at -20 ° C

Step 5: Preparation of 10% NP-40

• Mix 10 ml of NP-40 with 90 ml of ddH2O by stirring.
• Store at 4°C.

Table-1: Lysis buffer-cell

SL/No.	Store	Content	Stock	1 mL	2 mL	4 mL
1	RT	50 mM Tris-HCL (pH7.4)	1 M	50 µL	100 µL	200 µL
2	4	1% NP-40	10%	100 µL	200 µL	400 µL
3	4	0.25% Sodium Deoxycholate, NaDc	1%	250 µL	500 µL	1000µL
4	RT	150 mM NaCl	1 M	150 µL	300 µL	600 µL
5	-30	1 mM Na 3 VO 4 (Sodium orthovanadate)	200 mM	5 µL	10 µL	20 µL
6	RT	1 mM NaF (Sodium fluoride)	100 mM	10 µL	20 µL	40 µL
7	-30	1-2 mM protease inhibitor cocktail	× 7	143 µL	286 µL	571 µL
8	RT	D.W.		292 µL	584 µL	1169 µL

Table-2: Lysis buffer-tissue

Sl/No.	Store	Content	Stock	10 mL
1	RT	50 mM Tris-HCL (pH7.4)	1 M  (next, 100mM)	500 µL
2	RT	150 mM NaCl	1M (next, 300mM)	1.5 mL
3	RT	0.1% SDS	10%	100 µL
4	4	1% NP-40 (light protected)	10% in water (boiled 1ml/9ml)	1 mL
5	4	0.5% Sodium Deoxycholate, NaDc	10% in water (0.1 gram/1 mL)	500 µL
6	-20	1 mM Na 3 VO 4 (Sodium orthovanadate)	200 mM	50 µL
7	-20	5 mM NaF (Sodium fluoride)	500 mM (0.021 gram/1 mL)	100 µL
8	-20	protease inhibitor cocktail		1 tablet
9	RT	1 mM PMSF	200 mM in isopropanol (0.03484 gram/1 mL)	50 µL
10	RT	Autoclaved D.W.		6.2 mL

Note: PMSF use after 30 mins to dilution into buffer

• For clarifying stock solution: Click here
• For making working solution from stock: Click here

2. Buffer system

 A. 30％ Acrylamide solution

Content	Conc.	50 mL	100 mL	200 mL
Acrylamide	29%	14.5 g	29 g	58 g
Bis-acrylamide	1%	0.5 g	1 g	2 g
Total	30%	15 g	30 g	60 g

Protocol:

• It dissolves by applying heat.
• Preparation: beaker, filter paper, bottle
• Fill the beaker with foil and dissolve enough.
• Filtration with Whatman No.1 filter (0.45 µm)
• Put it in a brown bottle or a bottle filled with foil and
• store at 4 ℃ (1 month or less)

B. 1 M Tris-Cl (Lysis buffer pH 7.4 / Stacking gel pH 6.8)

• Autoclaved
• Molecular weight of Tris-Cl (MW: 121.14),

121.14 (g) x 1 M = 121.14 (g)

1000 mL: 121.14 = 200 mL : X

X= 121.14 ✖ 200/1000

X= 24.228 g

• S0, 24.228 g of Tris-Cl  dissolve in 180 mL of D.W. Adjust pH with HCl. Make the volume up to 200 mL.

C. 1.5 M Tris-Cl (running gel pH 8.8)

• Autoclaved
• Molecular weight of Tris-Cl (MW: 121.14),

121.14 (g) x 1.5 M = 181.71 (g)

1000 mL: 181.71 = 200 mL : X

X= 181.71 ✖ 200/1000

X= 36.342 g

• S0, 36.342 g of Tris-Cl  dissolve in 180 mL of D.W. Adjust pH with HCl. Make the volume up to 200 mL.

D. 10% SDS

If the total volume is 200 ml, add 20 (g). Ex) 200 ml × 10 (%) = 20 (g)

E. 10X SDS running buffer (pH 8.3)

Content	10X	1X
250 mM Tris-base	30.28 g	3.028 g
1.92 M glycine	144.13 g	14.41 g
1％ SDS	10 g	1 g
Total volume	1 L	1 L
Adjust the pH to 8.3 with HCl and adjust the total volume to 1L.

• To create 1X SDS running buffer immediately, calculate Con. of each buffer by 1/10.

• 25mM Tris base
• 192mM glycine
• 0.1％ SDS

F. 2× sample loading buffer (pH 6.8)

Content	1X	1X 10 mL	2X	2X 10 mL	20 mL
1M Tris-Cl (pH 6.8)	50 mM	1 mL	100 mM (0.1 M)	1 mL	2 mL
100％ Glycerol	10％	1 mL	20%	2 mL	4 mL
10％ SDS	2％	200 µL	4%	4 mL	8 mL
β-Mercaptoethanol	2.5％	250 µL	5%	500 µL	1 mL
4％ Bromophenol blue	0.1％	10 µL	0.2%	20 µL	1 mL
D.W.		7 mL 540 µL		2 mL 480 µL	4 mL

• Immediately prior to use, add β-Mercaptoethanol.
• Addition of 50 ul for 2X (because it is aliquoted by 1ml), addition of 25
• ul for 4X

G. 10× Transfer buffer (pH 8.3), 1L

Content	Qty.
Tris (250 mM)	30.28 g
Glycine (1920 mM)	140 g

Final volume 1L, then store at 4 ℃.

H. 1× Transfer buffer (pH 8.3), 1L

Content	Qty.
10X transfer buffer	100 mL
MetOH	200 mL
D.W.	700 mL

• MetOH must be added

I. 10× TBS (pH 7.5), 1L

Content	Qty.
NaCl (1.5 M)	87.66 g
Tris (100 mM)	12.114 g

• Finalize total volume 1L, then store by autoclave at 4 ℃.

J. 1× TBST, 1L

Content	Qty.
10X TBS	100 mL
D.W.	900 mL
Tween-20	1 mL

H. PBS (Phosphate buffer saline, pH7.4)

Content	Qty.
Working conc.	800 mL
137 mM NaCl	6.4 g
2.7 mM KCl	0.16 g
10 mM Na 2 HPO	1.152 g
2 mM KH 2 PO 4	0.192 g
D.W. up to 800 ml

I. Blocking buffer (5% skim milk in TBST)

• Two gels need 30 ml.
• When the blocking buffer is 30 ml, BSA (stored at -20 ° C) enters 300 μl

• Add BSA 1% along with desired quantity of TBST & skim milk.

TBST 500 mL + Skim milk 25 g → Vortexing

TBST 30 mL + Skim milk 1.5 g → Vortexing

TBST 20 mL + Skim milk 1 g → Vortexing