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구체적인 단백질 실험 프로토콜

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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

 

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