구체적인 단백질 실험 프로토콜

No7. Preparation of stock solution

Solution Method of preparation Comments

Phosphate-buffered saline (PBS) Dissolve 8.0g NaCl, 0.2g of KCl, 1.44g of  Na2HPO4, and 0.24g of KH2PO4 in 800ml of distilled H2O.

Adjust the pH to 7.4.
Adjust  the volume to 1L.
Dispense in convenient volumes and sterilize by autoclaving.

Store at room temperature. Can be made as a 10X stock.

Tris-buffered saline (TBS) (25mM Tris) Dissolve 8.0g of NaCl, 0.2g of KCl, and 3g of  Tris base in 800ml of distilled H2O.

Adjust the pH to 8.0 with 1M HCl.
Adjust the volume to 1 liter.
Dispense in convenient volumes and sterilize by  autoclaving.

Store at  room temperature.

10% Sodium azide Dissolve 10g of sodium azide in 100ml of distilled H2O.
Store at  room temperature. Do not use sodium azide for  experiments using live organisms or for reactions that
use  horseradish peroxidase.

3% Bovine serum albumin

in phosphate-buffered

saline (3% BSA/PBS) Add 3g of BSA (Fraction V) to 100ml of PBS.

Allow to dissolve.
Add o.2 ml of 10% sodium azide.

Store at 4°C.

1M Tris Dissolve 121g of Tris base in 800ml of distilled H2O.

Adjust to the desired pH by adding concentrated HCl.
Adjust the volume to 1000ml with distilled H2O.
Dispense in convenient volumes and sterilize by autoclaving.

Store at  room temperature.

500mM EDTA Add 186g of disodium ethylene diamine tetraacetate-2H2O

to 400ml of distilled H2O.
Add NaOH to adjust the pH to 8.0 and to allow the EDTA to dissolve.
Bring volume to   500ml with distilled H2O.
Dispense in convenient volumes and sterilize by autoclaving.

Store at  room temperature.

100% (wt/volume)

Trichloroacetic

acetic acid (TCA) Add 227ml of distilled H2O to a 500-g bottle of TCA

30% Acrylamide mix Dissolve 29.2g of acrylamide (electrophoresis frade)  and 0.8g of N, N'-methylene-bisacrylamide (electrophoresis grade) in 80ml of distilled H2O.

Adjust the volume to 100ml.

Store at room temperature.

1.5M Tris (pH 8.8) Dissolve 181.5g of Tris base in 800ml of distilled H2O.

Adjust the pH to 8.8 with concentrated HCl.
Adjust the volume to 1 liter.
Dispense in convenient volumes and sterilize by autoclaving.

Store at room temperature.

1.0M Tris (pH 6.8) Dissolve 12.1g of Tris base in 80ml of distilled H2O.

Adjust the pH to 6.8 with concentrated HCl.
Adjust the volume to 100 ml.
Dispense in convenient volumes and sterilize by autoclaving.

Store at room temperature.

10% Sodium dodecyl sulfate (SDS) Dissolve 10g of SDS in 100ml of distilled H2O.
Store at room temperature.

10% Ammonium persulfate (APS) Dissolve 0.5g of ammonium persulfate (electrophoresis grade) in 5ml of distilled H2O.
Store at 4°C. Make fresh solution weekly.

2X Laemmli sample buffer Add 4ml of 10% SDS, 2ml of glycerol, and 1.2ml of 1M Tris (pH6.8) to 2.8ml of distilled H2O.

Add 0.01% bromophenol blue as a tracking dye.

Store at room temperature.   To prepare 1X sampole buffer, mix 5 parts 2X, 4 parts water, and 1 part 1M DTT.

1M Dithiothreitol Dissolce 5g of DTT in 32ml of distilled H2O.

Dispense in 1-ml aliquots.

Store at -20°C.

10X Laemmli running buffer To a 10;liter carboy, add 8 liters of distilled H2O, 303g of Tris base, 1442g of glycine, and 100g of SDS.

After all the chemicals have dissolved, adjust the pH to 8.3.
Adjust tje vollume ro 10 liters with H2O.

Store at room temperature.

Destain To a 10-liter carboy add 2.5 liters of methanol and 700ml of glacial acetic acid.

Adjust the colume to 10 liters with H2O.

Store at room temperature.

4% Paraformaldehyde Dissolve EM grade paraformaldehyde in PBS in Pyrex container with stir bar (4g to 100ml)

Add a few drops of NaOH and heat in a hood (keep bottle cap loose_ at 60 °C to dissolve.
Cool to room temperature and adjust pH to 7.4.

Make fresh prior to use.

Gelvatol Dissolve 0.35g of Gelvatol in 3ml of deionized H2O and 1.5ml of glycerol. (PBS can be substituted for water)

Heat the solution with stirring in a boiling water bath until the gelvatol is completely dissolved.
Add anti-fade agents as desired.

Store at 4 °C  (can be stored for months).

Glycerol anti-fade mounting medium Dissolve in 100ml og glycerol: 5g of n-propyl gallate, 0.25g of DABCO, 2.5mg of p-phenylenediamine.

Add several pellets of NaOH to bring pH above neutral.
Stir thoroughly (>1day).

Store aliquots at -20°C wrapped in foil.

1M NaCL/ 0.05M sodium phosphate (pH7.5) For liter: Combine 200ml of 5M NaCl, 500 ml of 0.1M sodium phosphate(pH 7.5), and 300ml of H2O.

NP-40 lysis buffer For 1 liter: Combine 30 ml of 5M NaCl, 100ml of 10% NP-40, 50ml of 1M Tris (pH 8.0), and 820 ml of H2O.
Store at 4°C.

RIPA buffer For 1 liter: Combine 30 ml of 5M NaCl, 100ml of 10% NP-40, 50ml of DOC, 100ml of 10% SDS, 50ml of 1M Tris (pH 8.0), and 670ml of H2O.
Store at 4°C.

No8. Solutions for Tris/ glycine SDS-polyacrylamide gel electrophoresis

6% 5ml 10ml 15ml 20ml 25ml 30ml 40ml 50ml

H2O

30% Acrylamide mix
1.5M Tris (pH 8.8)
10% SDS
10% APS

TEMED 2.6

1.0
1.3
0.05
0.05

0.004 5.3

2.0
2.5
0.1
0.1

0.008 7.9

3.0
3.8
0.15
0.15

0.012 10.6

4.0
5.0
0.2
0.2

0.016 13.2

5.0
6.3
0.25
0.25

0.02 15.9

6.0
7.5
0.3
0.3

0.024 21.2

8.0
10.0
0.4
0.4

0.032 26.5

10.0
12.5
0.5
0.5

0.04

8% 5ml 10ml 15ml 20ml 25ml 30ml 40ml 50ml

H2O

30% Acrylamide mix
1.5M Tris (pH 8.8)
10% SDS
10% APS

TEMED 2,3

1.3
1.3
0.05
0.05

0.003 4.6

2.7
2.5
0.1
0.1

0.006 6.9

4.0
3.8
0.15
0.15

0.009 9.3

5.3
5.0
0.2
0.2

0.012 11.6

6.7
6.3
0.25
0.25

0.015 13.9

8.0
7.5
0.3
0.3

0.018 18.5

10.7
10.0
0.4
0.4

0.024 23.2

13.3
12.5
0.5
0.5

0.03

10% 5ml 10ml 15ml 20ml 25ml 30ml 40ml 50ml

H2O

30% Acrylamide mix
1.5M Tris (pH 8.8)
10% SDS
10% APS

TEMED 2.0

1.7
1.3
0.05
0.05

0.002 4.0

3.3
2.5
0.1
0.1

0.006 5.9

5
3.8
0.15
0.15

0.006 7.9

6.7
5.0
0.2
0.2

0.008 9.9

8.3
6.3
0.25
0.25

0.01 11.9

10.0
7.5
0.3
0.3

0.012 15.8

13.3
10.0
0.4
0.4

0.016 19.8

16.7
12.5
0.5
0.5

0.02

12% 5ml 10ml 15ml 20ml 25ml 30ml 40ml 50ml

H2O

30% Acrylamide mix
1.5M Tris (pH 8.8)
10% SDS
10% APS

TEMED 1.6

2.0
1.3
0.05
0.05

0.002 3.3

4.0
2.5
0.1
0.1

0.004 4.9

6.0
3.8
0.15
0.15

0.006 6.6

8.0
5.0
0.2
0.2

0.008 8.2

10.0
6.3
0.25
0.25

0.01 9.9

12.0
7.5
0.3
0.3

0.012 13.2

16.0
10.0
0.4
0.4

0.016 16.5

20.0
12.5
0.5
0.5

0.02

15% 5ml 10ml 15ml 20ml 25ml 30ml 40ml 50ml

H2O

30% Acrylamide mix
1.5M Tris (pH 8.8)
10% SDS
10% APS

TEMED 1.1

2.5
1.3
0.05
0.05

0.002 2.3

5.0
2.5
0.1
0.1

0.004 3.4

7.5
3.8
0.15
0.15

0.006 4.6

10.0
5.0
0.2
0.2

0.008 5.7

12.5
6.3
0.25
0.25

0.01 6.9

15.0
7.5
0.3
0.3

0.012 9.2

20.0
10.0
0.4
0.4

0.016 11.4

25.0
12.5
0.5
0.5

0.02

STACK 1ml 2ml 3ml 4ml 5ml 6ml 8ml 10ml

H2O

30% Acrylamide mix
1.0M Tris (pH 6.8)
10% SDS
10% APS

TEMED 0.68

0.17
0.13
0.01
0.01

0.001 1.4

0.33
0.25
0.02
0.02

0.002 2.1

0.5
0.38
0.03
0.03

0.003 2.7

0.67
0.5
0.04
0.04

0.004 3.4

0.83
0.63
0.05
0.05

0.005 4.1

1.0
0.75
0.06
0.06

0.006 5.5

1.3
1.0
0.08
0.08

0.008 6.8

1.7
1.25
0.1
0.1

0.01

구체적인 단백질 실험 프로토콜

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Solution	Method of preparation	Comments
Phosphate-buffered saline (PBS)	Dissolve 8.0g NaCl, 0.2g of KCl, 1.44g of Na2HPO4, and 0.24g of KH2PO4 in 800ml of distilled H2O. Adjust the pH to 7.4. Adjust the volume to 1L. Dispense in convenient volumes and sterilize by autoclaving. Store at room temperature.	Can be made as a 10X stock.
Tris-buffered saline (TBS) (25mM Tris)	Dissolve 8.0g of NaCl, 0.2g of KCl, and 3g of Tris base in 800ml of distilled H2O. Adjust the pH to 8.0 with 1M HCl. Adjust the volume to 1 liter. Dispense in convenient volumes and sterilize by autoclaving. Store at room temperature.
10% Sodium azide	Dissolve 10g of sodium azide in 100ml of distilled H2O. Store at room temperature.	Do not use sodium azide for experiments using live organisms or for reactions that use horseradish peroxidase.
3% Bovine serum albumin in phosphate-buffered saline (3% BSA/PBS)	Add 3g of BSA (Fraction V) to 100ml of PBS. Allow to dissolve. Add o.2 ml of 10% sodium azide. Store at 4°C.
1M Tris	Dissolve 121g of Tris base in 800ml of distilled H2O. Adjust to the desired pH by adding concentrated HCl. Adjust the volume to 1000ml with distilled H2O. Dispense in convenient volumes and sterilize by autoclaving. Store at room temperature.
500mM EDTA	Add 186g of disodium ethylene diamine tetraacetate-2H2O to 400ml of distilled H2O. Add NaOH to adjust the pH to 8.0 and to allow the EDTA to dissolve. Bring volume to 500ml with distilled H2O. Dispense in convenient volumes and sterilize by autoclaving. Store at room temperature.
100% (wt/volume) Trichloroacetic acetic acid (TCA)	Add 227ml of distilled H2O to a 500-g bottle of TCA
30% Acrylamide mix	Dissolve 29.2g of acrylamide (electrophoresis frade) and 0.8g of N, N'-methylene-bisacrylamide (electrophoresis grade) in 80ml of distilled H2O. Adjust the volume to 100ml. Store at room temperature.
1.5M Tris (pH 8.8)	Dissolve 181.5g of Tris base in 800ml of distilled H2O. Adjust the pH to 8.8 with concentrated HCl. Adjust the volume to 1 liter. Dispense in convenient volumes and sterilize by autoclaving. Store at room temperature.
1.0M Tris (pH 6.8)	Dissolve 12.1g of Tris base in 80ml of distilled H2O. Adjust the pH to 6.8 with concentrated HCl. Adjust the volume to 100 ml. Dispense in convenient volumes and sterilize by autoclaving. Store at room temperature.
10% Sodium dodecyl sulfate (SDS)	Dissolve 10g of SDS in 100ml of distilled H2O. Store at room temperature.
10% Ammonium persulfate (APS)	Dissolve 0.5g of ammonium persulfate (electrophoresis grade) in 5ml of distilled H2O. Store at 4°C.	Make fresh solution weekly.
2X Laemmli sample buffer	Add 4ml of 10% SDS, 2ml of glycerol, and 1.2ml of 1M Tris (pH6.8) to 2.8ml of distilled H2O. Add 0.01% bromophenol blue as a tracking dye. Store at room temperature.	To prepare 1X sampole buffer, mix 5 parts 2X, 4 parts water, and 1 part 1M DTT.
1M Dithiothreitol	Dissolce 5g of DTT in 32ml of distilled H2O. Dispense in 1-ml aliquots. Store at -20°C.
10X Laemmli running buffer	To a 10;liter carboy, add 8 liters of distilled H2O, 303g of Tris base, 1442g of glycine, and 100g of SDS. After all the chemicals have dissolved, adjust the pH to 8.3. Adjust tje vollume ro 10 liters with H2O. Store at room temperature.
Destain	To a 10-liter carboy add 2.5 liters of methanol and 700ml of glacial acetic acid. Adjust the colume to 10 liters with H2O. Store at room temperature.
4% Paraformaldehyde	Dissolve EM grade paraformaldehyde in PBS in Pyrex container with stir bar (4g to 100ml) Add a few drops of NaOH and heat in a hood (keep bottle cap loose_ at 60 °C to dissolve. Cool to room temperature and adjust pH to 7.4. Make fresh prior to use.
Gelvatol	Dissolve 0.35g of Gelvatol in 3ml of deionized H2O and 1.5ml of glycerol. (PBS can be substituted for water) Heat the solution with stirring in a boiling water bath until the gelvatol is completely dissolved. Add anti-fade agents as desired. Store at 4 °C (can be stored for months).
Glycerol anti-fade mounting medium	Dissolve in 100ml og glycerol: 5g of n-propyl gallate, 0.25g of DABCO, 2.5mg of p-phenylenediamine. Add several pellets of NaOH to bring pH above neutral. Stir thoroughly (>1day). Store aliquots at -20°C wrapped in foil.
1M NaCL/ 0.05M sodium phosphate (pH7.5)	For liter: Combine 200ml of 5M NaCl, 500 ml of 0.1M sodium phosphate(pH 7.5), and 300ml of H2O.
NP-40 lysis buffer	For 1 liter: Combine 30 ml of 5M NaCl, 100ml of 10% NP-40, 50ml of 1M Tris (pH 8.0), and 820 ml of H2O. Store at 4°C.
RIPA buffer	For 1 liter: Combine 30 ml of 5M NaCl, 100ml of 10% NP-40, 50ml of DOC, 100ml of 10% SDS, 50ml of 1M Tris (pH 8.0), and 670ml of H2O. Store at 4°C.

6%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O 30% Acrylamide mix 1.5M Tris (pH 8.8) 10% SDS 10% APS TEMED	2.6 1.0 1.3 0.05 0.05 0.004	5.3 2.0 2.5 0.1 0.1 0.008	7.9 3.0 3.8 0.15 0.15 0.012	10.6 4.0 5.0 0.2 0.2 0.016	13.2 5.0 6.3 0.25 0.25 0.02	15.9 6.0 7.5 0.3 0.3 0.024	21.2 8.0 10.0 0.4 0.4 0.032	26.5 10.0 12.5 0.5 0.5 0.04
8%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O 30% Acrylamide mix 1.5M Tris (pH 8.8) 10% SDS 10% APS TEMED	2,3 1.3 1.3 0.05 0.05 0.003	4.6 2.7 2.5 0.1 0.1 0.006	6.9 4.0 3.8 0.15 0.15 0.009	9.3 5.3 5.0 0.2 0.2 0.012	11.6 6.7 6.3 0.25 0.25 0.015	13.9 8.0 7.5 0.3 0.3 0.018	18.5 10.7 10.0 0.4 0.4 0.024	23.2 13.3 12.5 0.5 0.5 0.03
10%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O 30% Acrylamide mix 1.5M Tris (pH 8.8) 10% SDS 10% APS TEMED	2.0 1.7 1.3 0.05 0.05 0.002	4.0 3.3 2.5 0.1 0.1 0.006	5.9 5 3.8 0.15 0.15 0.006	7.9 6.7 5.0 0.2 0.2 0.008	9.9 8.3 6.3 0.25 0.25 0.01	11.9 10.0 7.5 0.3 0.3 0.012	15.8 13.3 10.0 0.4 0.4 0.016	19.8 16.7 12.5 0.5 0.5 0.02
12%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O 30% Acrylamide mix 1.5M Tris (pH 8.8) 10% SDS 10% APS TEMED	1.6 2.0 1.3 0.05 0.05 0.002	3.3 4.0 2.5 0.1 0.1 0.004	4.9 6.0 3.8 0.15 0.15 0.006	6.6 8.0 5.0 0.2 0.2 0.008	8.2 10.0 6.3 0.25 0.25 0.01	9.9 12.0 7.5 0.3 0.3 0.012	13.2 16.0 10.0 0.4 0.4 0.016	16.5 20.0 12.5 0.5 0.5 0.02
15%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O 30% Acrylamide mix 1.5M Tris (pH 8.8) 10% SDS 10% APS TEMED	1.1 2.5 1.3 0.05 0.05 0.002	2.3 5.0 2.5 0.1 0.1 0.004	3.4 7.5 3.8 0.15 0.15 0.006	4.6 10.0 5.0 0.2 0.2 0.008	5.7 12.5 6.3 0.25 0.25 0.01	6.9 15.0 7.5 0.3 0.3 0.012	9.2 20.0 10.0 0.4 0.4 0.016	11.4 25.0 12.5 0.5 0.5 0.02
STACK	1ml	2ml	3ml	4ml	5ml	6ml	8ml	10ml
H2O 30% Acrylamide mix 1.0M Tris (pH 6.8) 10% SDS 10% APS TEMED	0.68 0.17 0.13 0.01 0.01 0.001	1.4 0.33 0.25 0.02 0.02 0.002	2.1 0.5 0.38 0.03 0.03 0.003	2.7 0.67 0.5 0.04 0.04 0.004	3.4 0.83 0.63 0.05 0.05 0.005	4.1 1.0 0.75 0.06 0.06 0.006	5.5 1.3 1.0 0.08 0.08 0.008	6.8 1.7 1.25 0.1 0.1 0.01