3. Pepsin from porcine gastric mucosa; powder !400 U/mg
protein.
4. Fetal bovine serum (FBS), 20%, heat inactivated, sterilefiltered.
5. RPMI 1640 culture medium with L-glutamine and sodium
bicarbonate; liquid, sterile-filtered.
6. Culture medium working solution: for 20 mL of culture
medium, use 16 mL of RPMI 1640 culture medium, 4 mL of
FBS, and 0.2 g of pepsin. Mix and adjust pH ¼ 4 with HCl (see
Note 1).
7. Restriction endonuclease Hinf I with buffer B 10Â in the set.
2.3 Protein
Extraction
and Precipitation
1. Lysis buffer: 60 mM Tris–HCl pH 7.5, 1% lauryl-maltoside,
5 mM phenylmethylsulfonyl fluoride (PMSF), and 1% dithiothreitol (DTT). Add 15 mL Tris solution (0.7268 g of Tris
dissolve in 100 mL of water) to a 50 mL graduated cylinder or a
glass beaker. Weigh 0.2 g of lauryl-maltoside, 0.308 g of DTT,
and 0.017 g of PMSF and transfer to the glass beaker. Mix and
adjust pH with HCl. Make up to 20 mL with water. Store at
4
C.
2. Protein quantification assay (e.g. BCA protein assay).
3. 99.9% Methanol.
4. 99.9% Chloroform.
2.4 SDSPolyacrylamide Gel
Electrophoresis
1. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Add about
100 mL water to a 500 mL graduated cylinder or a glass beaker
(see Note 2). Weigh 90.86 g Tris and transfer to the glass
beaker. Add water to a volume of 450 mL. Mix and adjust
pH with HCl (see Note 1). Make up to 500 mL with water.
Store at 4
C.
2. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Add about
20 mL water to a 100 mL graduated cylinder or a glass beaker.
Weigh 6.057 g Tris and transfer to the glass beaker. Add water
to a volume of 70 mL. Mix and adjust pH with HCl (see Note
2). Make up to 100 mL with water. Store at 4
C.
3. Thirty percent acrylamide/Bis solution (29:1). Weigh 29 g of
acrylamide monomer and 1 g Bis (cross-linker) and transfer to
a 100 mL graduated glass beaker containing about 40 mL of
water. Mix on a magnetic stirrer for 30 min. Make up to
100 mL with water and filter through a 0.45 μm Corning filter
(see Note 3). Store at 4
C, in a bottle wrapped with an
aluminum foil (see Note 4).
4. Ammonium persulfate (APS): 10% solution in water; weight
1 g and transfer to 1 mL Eppendorf tube containing 1 mL of
water (see Note 5).
5. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED) (see Note 6).
62
Robert Stryin ´ ski et al.
protein.
4. Fetal bovine serum (FBS), 20%, heat inactivated, sterilefiltered.
5. RPMI 1640 culture medium with L-glutamine and sodium
bicarbonate; liquid, sterile-filtered.
6. Culture medium working solution: for 20 mL of culture
medium, use 16 mL of RPMI 1640 culture medium, 4 mL of
FBS, and 0.2 g of pepsin. Mix and adjust pH ¼ 4 with HCl (see
Note 1).
7. Restriction endonuclease Hinf I with buffer B 10Â in the set.
2.3 Protein
Extraction
and Precipitation
1. Lysis buffer: 60 mM Tris–HCl pH 7.5, 1% lauryl-maltoside,
5 mM phenylmethylsulfonyl fluoride (PMSF), and 1% dithiothreitol (DTT). Add 15 mL Tris solution (0.7268 g of Tris
dissolve in 100 mL of water) to a 50 mL graduated cylinder or a
glass beaker. Weigh 0.2 g of lauryl-maltoside, 0.308 g of DTT,
and 0.017 g of PMSF and transfer to the glass beaker. Mix and
adjust pH with HCl. Make up to 20 mL with water. Store at
4
C.
2. Protein quantification assay (e.g. BCA protein assay).
3. 99.9% Methanol.
4. 99.9% Chloroform.
2.4 SDSPolyacrylamide Gel
Electrophoresis
1. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Add about
100 mL water to a 500 mL graduated cylinder or a glass beaker
(see Note 2). Weigh 90.86 g Tris and transfer to the glass
beaker. Add water to a volume of 450 mL. Mix and adjust
pH with HCl (see Note 1). Make up to 500 mL with water.
Store at 4
C.
2. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Add about
20 mL water to a 100 mL graduated cylinder or a glass beaker.
Weigh 6.057 g Tris and transfer to the glass beaker. Add water
to a volume of 70 mL. Mix and adjust pH with HCl (see Note
2). Make up to 100 mL with water. Store at 4
C.
3. Thirty percent acrylamide/Bis solution (29:1). Weigh 29 g of
acrylamide monomer and 1 g Bis (cross-linker) and transfer to
a 100 mL graduated glass beaker containing about 40 mL of
water. Mix on a magnetic stirrer for 30 min. Make up to
100 mL with water and filter through a 0.45 μm Corning filter
(see Note 3). Store at 4
C, in a bottle wrapped with an
aluminum foil (see Note 4).
4. Ammonium persulfate (APS): 10% solution in water; weight
1 g and transfer to 1 mL Eppendorf tube containing 1 mL of
water (see Note 5).
5. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED) (see Note 6).
62
Robert Stryin ´ ski et al.