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

 

No9. David's life chart 2

Molecular  Weight (daltons)	1 ug	1 nmole
100	10 nmoles or   6*10의 15제곱 molecules	0.1 ug
1,000	1 nmole or  6*10의 14제곱 molecules	1 ug
10,000	100 pmoles or  6*10의 13제곱 molecules	10 ug
20,000	50 pmoles or  3*10의 13제곱 molecules	20 ug
30,000	33 pmoles or  2*10의 13제곱 molecules	30 ug
40,000	25 pmole or   1.5*10의 13제곱 molecules	40 ug
50,000	20 pmoles or  1.2*10의 13제곱 molecules	50 ug
60,000	17 pmoles or  10의 13제곱 molecules	60 ug
70,000	14 pmoles or   8.6*10의 12제곱 molecules	70 ug
80,000	12 pomoles or  7.5*10의 12제곱 molecules	80 ug
90,000	11 pmoles or  6.6*10의 12제곱 molecules	90 ug
100,000	10 pmoles or  6*10의 12제곱 molecules	100 ug
120,000	8.3 pmoles or  5*10의 12제곱 molecules	120 ug
140,000	7.1 pmoles or  4.3*10의 12제곱 molecules	140 ug
160,000	6.3 pmoles or  3.8*10의 12제곱 molecules	160 ug
180,000	5.6 pmoles or  3.3*10의 12제곱 molecules	180 ug
200,000	5 pmoles or  3*10의 12제곱 molecules	200 ug

David's life chart 3

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 Some useful nucleotide dimensions

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 1cm of DNA ≈3 * 10의 6제곱 nucleotides

Organism	Base pairs/  haploid genome	Base pairs/  diploid genome	Length / cell	Mass
Human	3 * 10의 9제곱	6 * 10의 9제곱	2 meters (diploid)	6 pg
Fly	1.65 * 10의 8제곱	3.3 * 10의 8제곱	100 cm (diploid)	0.3 pg
Yeast	1.35 * 10의 7제곱	2.7 * 10의 7제곱	10 cm (diploid)	0.03 pg
E.coli	4.7 * 10의 6제곱	-	1.5 cm (haploid)	0.0045 pg
SV40	5 * 10의 3제곱	-	1.7 nm	0.000006 pg

Some useful cell dimensions

Organism	Dimensions	Volume
S.cerevisiae	5 um	66 um³
S. pombe	2 * 7 um	22 um³
Mammalian cell	10-20 um	500-4,000 um³
E.coli	1 * 3 um	2 um³
Mammalian mitochondrion	1 um	0.5 um³
Mammalian nucleus	5-10 um	66-500 um³
Plant chloroplast	1 * 4 um	3 um³
Bacteriophage lambda	50 nm (head only)	6.6 * 10의 -5제곱 um³
Ribosome	30 nm diameter	1.4 * 10의 -5제곱 um³
Globular monomeric protein	5 nm diameter	6.6 * 10의 -8제곱 um³

Some useful concentrarions

Total cell protein concentrations	Detergent soluble protein = 1-2 mg/10의 7제곱 mammalian  cells or 100 -200 mg/ml for soluble proteins only
Specific protein concentrations
Nucleus (200 um³):  Abundant transcription factor  Rare transcription factor	1nM (100,000 copies / nucleus)  10 pM (1,000 copies / nucleus)
Serum	50 - 100 mg / ml