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APPENDICES
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and Western Blotting
Appendix A1: SDS-PAGE Solutions
1. 2X Laemmli sample buffer (10 ml)
Tris-HCl 1 M (pH 6.8), BPB dye
Glycerol
SDS 10%
dH2O
(NOTE: Aliquot and store at -20°C)
2.5 ml
2 ml
4 ml
1.5 ml
2. 2X Laemmli sample buffer (with 2ME)
2X Laemmli sample buffer
2-Mercaptoethanol (2ME)
(NOTE: Aliquot and store at -20°C)
950 µl
50 µl
3. Monomer stock solution (30% Acryl A, 0.8% Bis)
Acrylamide
Bis
4. SDS 10% (100 ml)
Sodium dodecyl sulfate (SDS)
dH2O added to make 100 ml
5. Initiator (APS 2.5%)
Ammonium persulfate (APS)
dH2O
6. Electrophoresis running buffer (10X stock) (1 L)
Tris 0.25 M
Glycine 1.92 M
dH2O added to make 1 L
7. Electrophoresis running buffer (1X) (500 ml)
Electrophoresis running buffer 10X stock
SDS 10%
dH2O
97
60 g
1.6 g
10 g
0.025 g
1 ml
30.29 g
144.1 g
50 mL
5 mL
445 mL
Optimized SDS gel preparation
Solutions
30% Acryl A, 0.8% Bis
Tris-HCl 0.75 M, pH 8.8
Tris-HCl 1 M, BPB dye, pH 6.8
Ammonium persulfate (APS) 2.5%
dH2O
Total volume before TEMED
TEMED
10% Resolving gel
(µl)
2500
3750
240
1010
7500
4% Stacking gel
(µl)
400
3750
35
2190
3000
15
1. Prepare resolving gel first by mixing all solutions in a small beaker/50
ml Falcon tube. Keep the mixture on ice until ready to use. Add
TEMED only when ready to use because the mixture will immediately
start to solidify as soon as TEMED is added due to it being a
solidifying agent.
2. Pipette the resolving gel mixture in between the glass cassettes.
Immediately add a layer of dH2O (1 ml) on top of the resolving gel to
ensure the gel is straight. Leave the gel to solidify (15 – 30 min).
3. After solidifying, remove the dH2O layer by tipping it over and using
a filter paper. Put in a plastic comb between the glass cassettes above
the solidified resolving gel.
4. Prepare the stacking gel mixture and slowly pipette it in. Make sure
there are no air bubbles. Leave it to solidify (10 – 15 min).
5. After solidifying, carefully remove the comb and immerse the gel in
electrophoresis running buffer 1X. Gel is ready to use.
Appendix A2: Protein electrotransfer
1. Transfer buffer (1 L)
Electrophoresis running buffer (10X stock)
Tris 0.75 M (9.085 g + dH2O to make 100 ml)
Methanol (10% of total solution)
SDS 10%
dH2O to make 1 L
100 ml
100 ml
100 ml
2 ml
698 ml
Assembly
1. Cut 8 pieces of filter paper (6 cm x 5 cm).
2. Cut 1 piece of PVDF membrane (6 cm x 6 cm).
3. Using forceps, activate the PVDF membrane in methanol for 10
seconds. Be sure to wear gloves. Keep the activated PVDF membrane
in transfer buffer until ready to use.
4. Submerge the filter paper pieces in transfer buffer for at least 5 min
before use.
5. Stack:
• 4 filter paper ® PVDF membrane ® SDS gel ® 4 filter paper
98
Pour some transfer buffer in between every layer to keep
everything wet.
Remove air bubbles every layer using a roller.
Appendix A3: Western Blotting Solutions
1. Phosphate buffer saline (PBS) (25X stock) (1 L)
Sodium chloride (NaCl)
Potassium chloride (KCl)
NA2HPO4
(disodium phosphate/sodium hydrogenphosphate)
KH2PO4
200 g
5g
36 g
5g
(monopotassium phosphate/potassium dihydrogen phosphate)
Dissolve in ~800 ml dH2O
Adjust pH to 7.4 with HCl/NaOH
Add dH2O to make 1 L
2. Phosphate buffer saline (PBS) (1X) (500 ml)
PBS (25X stock)
dH2O
3. PBS/T (PBS with 0.05% Tween-20)
PBS (1X)
Tween-20
Mix well before use
20 ml
480 ml
1L
0.5 ml
4. Blocking buffer (PBS + 5% BSA)
Skim milk
PBS (1X)
0.5 g
10 ml
5. PBS + 1% BSA
Skim milk
PBS (1X)
0.1 g
10 ml
Appendix A4: Protein Purification Solutions
1. Sodium azide (NaN3) (10% stock) (50 ml)
Sodium azide (NaN3)
Add dH2O to make 50 ml
5g
2. Tris-buffer saline (TBS) (1X) + NaN3 0.02% (500 ml)
Tris
3.025 g
Sodium chloride (NaCl)
4.39 g
Add dH2O to make 500 ml
NaN3 10% stock
1 ml
99
3. Acidic buffer: Gly-HCl 0.1 M, pH 2.5 (40 ml)
Glycine
dH2O
Adjust to pH 2.5 with HCl
Add dH2O to make 40 ml
4. Basic buffer: Tris-HCl 1 M, pH 9 (40 ml)
Tris
dH2O
Adjust to pH 9 with HCl
Add dH2O to make 40 ml
5. NaHCO3 0.1 M, NaCl 0.5 M, pH 8.3 (100 ml)
Sodium hydrogencarbonate (NaHCO3)
Sodium chloride (NaCl)
dH2O
Adjust to pH 8.3
Add dH2O to make 100 ml
0.3 g
30 ml
4.85 g
30 ml
0.84 g
2.922 g
80 ml
6. Phenylmethylsulfonyl fluoride (PMSF) (100 mM stock) (1 ml)
Phenylmethylsulfonyl fluoride (PMSF)
0.0174 g
2-propanol
1 ml
7. Phenylmethylsulfonyl fluoride (PMSF) 0.5 mM in a 10 ml-solution
PMSF (100 mM stock)
50 µl
Solution
10 ml
8. AESBF (Pefabloc) (100 mM stock) (1 ml)
Pefabloc
dH2O
0.024 g
1 ml
9. AESBF (Pefabloc) 2 mM in a 10 ml-solution
Pefabloc (100 mM stock)
0.2 ml
10. AESBF (Pefabloc) 0.5 mM in a solution
Pefabloc (100 mM stock)
50 µl
11. TBS + EDTA 10 mM (50 ml)
EDTA
TBS 1X
0.186 g
50 ml
12. Starting buffer (SB), Tris-HCl 50 mM, NaCl 200 mM, CaCl2 10 mM,
pH 7.8 (500 ml)
Tris-HCl (1 M stock)
25 ml
Sodium chloride (NaCl)
5.844 g
Calcium chloride (CaCl2)
0.735 g
dH2O
480 ml
100
Adjust to pH 7.8
Add dH2O to make 500 ml
13. Methyl-a-mannopyranoside (MaM) in SB (300 mM stock) (30 ml)
Methyl-a-mannopyranoside (MaM)
1.748 g
Starting buffer (SB)
30 ml
14. Methyl-a-mannopyranoside (MaM) in SB (50 mM) (10 ml)
MaM (300 mM stock)
1.67 ml
Starting buffer (SB)
8.33 ml
15. Methyl-a-mannopyranoside (MaM) in SB (5 mM) (10 ml)
MaM (50 mM)
1 ml
Starting buffer (SB)
9 ml
16. EDTA 10 mM in PBS (10 ml)
EDTA [2NA (EDTA.2Na) Dodite]
PBS
0.037 g
10 ml
Appendix A4: Protein Expression and Purification
1. Lysozyme (10 mg/ml stock)
Lysozyme
dH2O
0.01 g
1 ml
2. Lysis buffer (1% TX100, TBS, lysozyme 100 µg/ml)
Triton X-100 (TX100)
TBS 1X
Lysozyme (10 mg/ml stock)
0.2 ml
19.8 ml
0.2 ml
3. Sodium phosphate buffer (NaPB) (200 mM stock) (50 ml)
NaH2PO4–H2O
1.38 g
(Sodium phosphate monobasic monohydrate)
Na2HPO4
1.42 g
(Disodium phosphate)
Add dH2O to make 50 ml, adjusted to pH 7.2
4. Sodium chloride (NaCl) (3 M stock) (50 ml)
NaCl
Add dH2O to make 50 ml
8.77 g
5. Imidazole (1 M stock) (50 ml)
Imidazole
Add dH2O to make 50 ml
3.4 g
6. Binding buffer (NaPB 20 mM, NaCl 300 mM, Imidazole 10 mM) (50
ml)
101
NaPB (200 mM stock)
NaCl (3 M stock)
Imidazole (1 M stock)
dH2O
5 ml
5 ml
0.5 ml
39.5 ml
7. Wash buffer I (NaPB 20 mM, NaCl 300 mM, Imidazole 20 mM) (50
ml)
NaPB (200 mM stock)
5 ml
NaCl (3 M stock)
5 ml
Imidazole (1 M stock)
1 ml
dH2O
39 ml
8. Wash buffer II (NaPB 20 mM, NaCl 300 mM, Imidazole 50 mM) (50
ml)
NaPB (200 mM stock)
5 ml
NaCl (3 M stock)
5 ml
Imidazole (1 M stock)
2.5 ml
dH2O
37.5 ml
9. Elution buffer (NaPB 20 mM, NaCl 300 mM, Imidazole 250 mM) (50
ml)
NaPB (200 mM stock)
5 ml
NaCl (3 M stock)
5 ml
Imidazole (1 M stock)
12.5 ml
dH2O
27.5 ml
102
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