Stock Solutions
Coomassie Blue (~1 liter)
Destain (10.2 liter)
(add in this sequence)
Coommassie blue R250 (brilliant blue) 1 g
Methanol
270 ml
dH2O
630 ml
37% Formaldehyde
150 ml
Coomassie 1:40 for microbicidal assays:
Same as above but use only 25 mg/1 l of the dye
Methanol
dH2O
37% Formaldehyde
5% HoAc (4 liter)
5 M NaCl (1 liter)
2.5 l
7.5 l
200 ml
(add in this sequence)
dH2O
Acetic acid, glacial
3,800 ml
200 ml
NaCl
dH2O
ad
292.2 g
1,000 ml
(Ad means “fill up to”)
200 mM NaH2PO4 monobasic
200 mM Na2HPO4 dibasic
For 200 mM measure 1/5th of the molecular weight on
the container/ per liter desired end volume.
Remember to add water to the 1 liter mark and do not
add 1 liter of water to the dry salt.
The amount weighed out depends on the hydration of the
salt in the package!
For 200 mM measure 1/5th of the molecular weight
on the container/ per liter desired end volume.
Remember to add water to the 1 liter mark and do
not add 1 liter of water to the dry salt.
The amount weighed out depends on the hydration
of the salt in the package!
Sodium phosphate buffers
Prepare mono and dibasic stock solutions at the desired
molarity. Make sure both stocks have the same molarity!
Then mix the two until the desired pH is achieved.
Monobasic is acidic and dibasic is alkaline. Below is a
table how to mix the two stock solutions for selected pH
values.
pH
5.7
6.0
6.5
7.0
7.4
8.0
% mono basic + % dibasic
93.5% + 6.5%
87.7% + 12.3%
68.5% + 31.5%
39% + 61%
19% + 81%
5.3% + 94.7%
10X PBS (1 liter)
10X Blotto PBS (1 liter)
KCl
KH2PO4 (unhydrated)
NaCl
Na2HPO4.7H2O
dH2O
for 500 ml 200 mM sodium phosphate buffer pH
7.4 mix as follows:
200 mM dibasic
405 ml
200 mM monobasic
95 ml
sterile filter
2g
2g
80 g
21.6 g
ad 1000 ml
(Ad means “fill up to”)
Used to make 1X blotto PBS for blocking and
antibody diluting solution.
200 mM Na phosphate buffer pH 7.4 500 ml
NaCl
90 g
let it dissolve
dH2O
ad 1000 ml
(Ad means “fill up to”)
Porter Lab revised 1/27/09
1
Stock Solutions
1 M Tris (1 liter, pH 7.5)
10X Blotto TBS (1 liter, low salt)
Tris-HCl
Tris-Base
H2O
127 g
23 g
800 ml
Adjust to pH 7.5 with HCl or NaOH
Used to make 1X blotto TBS for BSA-wash in
western protocol
NaCl
90 g
dH2O
700 ml
Tris HCl (Trizma hydrochloride) 31.52 g
dH2O
pH to 7.4 with HCl or NaOH
ad 1000 ml
(Ad means “fill up to”)
dH2O
ad 1000 ml
(Ad means “fill up to”)
5X TBS (1 liter, high salt)
0.75% Blotto (100 ml)
Tris buffered saline.
Non-fat Milk powder (e.g. carnation)
1 X Blotto PBS
1M Tris buffer pH 7.5
NaCl
dH2O
100 ml
146.1 g
ad 1000 ml
0.75 g
100 ml
keep at °4C
(Ad means “fill up to”)
1M NH4Ac (1 liter)
Ammonium acetate
dH2O
AP Buffer (500 ml)
77.08 g
ad 1000 ml
100 mM NaCl/ 5 mM MgCl2/ 100 mM Tris base, pH
9.8
(Ad means “fill up to”)
10% Ammonium Persulfate (APS, 10 ml)
APS
dH2O
1g
10 ml
5 M NaCl
1 M MgCl2
Tris Base
H2O
pH to 9.8 with HCl or NaOH
dH2O
10 ml
2.5 ml
6.055 g
400 ml
ad 500 ml
(Ad means “fill up to”)
10% SDS (250 ml)
Nitro Blue Tetrazolium (NBT)
dH20
Lauryl sulfate
225 ml
25 g
Wear mask to weigh. Weigh under fume hood.
If it is NBT sodium salt: dissolve in water!!!
If not: use 70% DMF to dissolve
NBT (in freezer)
70% DMF
1g
20 ml
Gel Drying Solution (1 liter)
Glycerol
Ethanol (Reagent alcohol)
dH2O
50 ml
200 ml
ad 1000 ml
Light sensitive
Wrap in foil and place in 4ºC refrigerator
(Ad means “fill up to”)
5-Bromo-4-Chloro-3-indolyl phosphate (BCIP)
70% Dimethylformamide (DMF)
BCIP (in freezer)
100 mg
100% DMF
2 ml
Light sensitive. Wrap in foil and place in 4ºC
dH2O
DMF (found in flammable)
keep in flammable cabinet
30 ml
70 ml
refrigerator
Porter Lab revised 1/27/09
2
Stock Solutions
TRICINE SDS-PAGE
Gel Pouring
5 x Laemli Buffer
For 4 mini gels:
Tris base
Glycine
dH2O
SDS
Separating gel
Glyercol
ddH2O
SDS-Gel buffer
Acrylamide solution
10% Ammonium persulfate (APS)
TEMED
6.58 ml
13.6 ml
16 ml
12 ml
240 l
24 l
Stacking gel
ddH2O
SDS-Gel buffer
Acrylamide solution
10% Ammonium persulfate
TEMED
13.4 ml
4.96 ml
1.6 ml
160 l
16 l
1.51 g
7.22 g
ad 100 ml
0.5 g
Dilute 1 : 5 to make 1X Laemli buffer
3X SDS Loading buffer
1 M Tris pH 8.8
10% SDS
Glycerol
Mix
Add Bromophenol blue
5 ml
18 ml
7 ml
1 grain
Cathode Buffer (1 liter)
Upper buffer, 0.1 M tricine/0.1 M Tris/ 0.1% SDS,
pH 8.25
Acrylamide Solution
dH2O
Acrylamide
Bis-acrylamide
Let dissolve (will take time!)
dH2O
30 ml
48.5 g
1.5 g
ad
100 ml
Tricine
17.9 g
Tris base
12.1 g
dH2O
800 ml
Adjust pH to 8.25 with NaOH or HCl
dH2O
ad 1000 ml
SDS
1g
Wrap bottle in foil.
Gel Buffer (1 liter)
Wear mask for SDS. Weigh out SDS under the fume
hood.
3 M Tris base/ 0.3% SDS/ pH 8.4
Anode Buffer (1 liter)
Tris Base
363.3 g
dH2O
500 ml
Let dissolve
Adjust pH to 8.4 with NaOH or HCl
dH2O
ad 1000 ml
SDS (sodium lauryl sulfate)
3 g
Lower buffer, 0.2 M Tris, pH 8.9
Wear mask when weighing SDS. Weigh under the fume
hood.
SDS- Transfer buffer (3 liters)
dH2O
100% methanol
0.5 M Borate [pH 9.0]*
Mix
10% SDS
Tris base
24.2 g
dH2O
800 ml
Adjust pH to 8.25 with NaOH or HCl
dH2O
ad 1000 ml
2085 ml
600 ml
300 ml
15 ml
* adjust with NaOH
(Ad means “fill up to”)
Porter Lab revised 1/27/09
3
Stock Solutions
AU-PAGE
Gel Pouring
3X AU Sample buffer
For 10 mini gels:
9 M urea in 5% acetic acid with methyl green
Urea
dH2O
Solution A
Solution B
10% Ammonium persulfate (APS)
28.8 g
40.5 ml
20.1 ml
12 ml
1.8 ml
Urea
dH2O
deionize with resin
Glacial acetic acid
dH2O
27 g
~ 20 ml
ad
2.5 ml
50 ml
Solution A
Add a tiny bit of methyl green to yield a middle-dark
green/blue color
60 % acrylamide/1.6% bisacrylamide
dH2O
Acrylamide
Bis-acrylamide
Let dissolve (will take time!)
dH2O
150 ml
120 g
3.2 g
ad
Aliquot per 1.5 ml and keep at -20°C.
200 ml
AU- Transfer buffer (3 liters)
dH2O
100% methanol
Glacial acetic acid
Wrap bottle in foil.
Solution B
2679 ml
300 ml
21 ml
43.2 % acetic acid/4% TEMED (vol/vol)
Glacial acetic acid
TEMED
dH2O
21.6 ml
2 ml
26.4 ml
Note: this will produce heat and fumes.
Wrap bottle in foil.
Porter Lab revised 1/27/09
4