Laboratory Methods in Enzymology: Protein Part C (eBook)

eBook Download: PDF | EPUB

2014 | 1. Auflage
296 Seiten
Elsevier Science (Verlag)
978-0-12-420178-1 (ISBN)

In this volume we have brought together a number of core protocols concentrating on Protein, carefully written and edited by experts. - Indispensable tool for the researcher - Carefully written and edited by experts to contain step-by-step protocols - In this volume we have brought together a number of core protocols concentrating on Protein

In this volume we have brought together a number of core protocols concentrating on Protein, carefully written and edited by experts. - Indispensable tool for the researcher- Carefully written and edited by experts to contain step-by-step protocols- In this volume we have brought together a number of core protocols concentrating on Protein

Front Cover 1
Laboratory Methods in Enzymology: Protein Part C 4
Copyright 5
Contents 6
Contributors 14
Miscellaneous 16
Preface 18
Section I: Protein Protocols/Protein Precipitation 20
Chapter One: TCA Precipitation 22
1. Theory 23
2. Equipment 23
3. Materials 23
3.1. Solutions and buffers 24
4. Protocol 25
4.1. Preparation 25
4.2. Duration 25
4.3. Caution 25
5. Step 1A Trichloroacetic Acid Precipitation 25
5.1. Overview 25
5.2. Duration 25
5.3. Tip 26
5.4. Tip 26
5.5. Tip 26
6. Step 1B Deoxycholate-Trichloroacetic Acid Precipitation 26
6.1. Overview 26
6.2. Duration 26
6.3. Tip 27
6.4. Tip 28
6.5. Tip 28
References 29
Referenced Protocols in Methods Navigator 29
Section II: Protein Protocols/Protein Pull-Down Methods 30
Chapter Two: Coimmunoprecipitation of Proteins from Yeast 32
1. Theory 33
2. Equipment 34
3. Materials 34
3.1. Solutions and buffers 35
4. Protocol 36
4.1. Duration 36
4.2. Preparation 36
5. Step 1 Preparation of Whole Cell Lysates 37
5.1. Overview 37
5.2. Duration 37
6. Step 2 Normalization of Cell Lysates 39
6.1. Overview 39
6.2. Duration 39
6.3. Tip 39
7. Step 3 Coimmunoprecipitation 39
7.1. Overview 39
7.2. Duration 40
7.3. Tip 41
7.4. Tip 41
7.5. Tip 41
7.6. Tip 41
7.7. Tip 41
7.8. Tip 41
7.9. Note 41
8. Step 4 Wash and Elute the Immunoprecipitates 42
8.1. Overview 42
8.2. Duration 42
8.3. Tip 43
8.4. Tip 43
9. Step 5 Analysis of Immunoprecipitations 43
9.1. Overview 43
9.2. Duration 44
References 45
Referenced Literature 45
Related Literature 45
Referenced Protocols in Methods Navigator 45
Chapter Three: Coupling Antibody to Cyanogen Bromide-Activated Sepharose 46
1. Theory 47
2. Equipment 47
3. Materials 47
3.1. Solutions and buffers 47
4. Protocol 49
4.1. Duration 49
4.2. Preparation 49
5. Step 1 Preparation of Antibody and Resin 49
5.1. Overview 49
5.2. Duration 49
5.3. Tip 50
5.4. Tip 50
5.5. Tip 50
5.6. Tip 50
6. Step 2 Coupling the Antibody to the Resin 51
6.1. Overview 51
6.2. Duration 51
6.3. Tip 51
7. Step 3 Quench the Reaction 51
7.1. Overview 51
7.2. Duration 51
7.3. Tip 52
8. Step 4 Wash the Resin 52
8.1. Overview 52
8.2. Duration 53
References 53
Referenced Protocols in Methods Navigator 53
Chapter Four: Analysis of Protein-Protein Interactions by Coimmunoprecipitation 54
1. Theory 55
2. Equipment 57
3. Materials 58
3.1. Solutions and buffers 59
4. Protocol 61
4.1. Preparation 61
4.2. Duration 61
5. Step 1 Isolation of the Protein of Interest by Immunoprecipitation 61
5.1. Overview 61
5.2. Duration 62
5.3. Tip 62
5.4. Tip 62
5.5. Tip 62
6. Step 2 Detection of the Binding Partner by Immunoblotting 63
6.1. Overview 63
6.2. Duration 63
6.3. Tip 64
6.4. Tip 64
References 65
Referenced Literature 65
Referenced Protocols in Methods Navigator 66
Section III: Protein Protocols/Protein Purification 68
Chapter Five: Use and Application of Hydrophobic Interaction Chromatography for Protein Purification 70
1. Theory 71
1.1. Latest technology in HIC adsorbents 73
1.2. Advantages and disadvantages of using HIC 74
2. Equipment 75
3. Materials 75
3.1. Solutions and buffers 76
3.2. Preparation 76
4. Protocol 77
4.1. Preparation 77
4.2. Duration 77
5. Step 1 Column Equilibration 78
5.1. Overview 78
5.2. Duration 79
6. Step 2 Column Loading 79
6.1. Overview 79
6.2. Duration 79
7. Step 3 Product Elution 80
7.1. Overview 80
7.2. Duration 80
7.3. Gradient elution 81
7.4. Stepwise (isocratic) elution 82
8. Step 4 Adsorbent Regeneration and Sanitization 82
8.1. Overview 82
8.2. Duration 82
References 83
Referenced Literature 83
Source References 84
Referenced Protocols in Methods Navigator 84
Chapter Six: Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins 86
1. Theory 87
2. Equipment 88
3. Materials 88
3.1. Solutions and buffers 89
4. Protocol 91
4.1. Duration 91
4.2. Preparation 91
4.2.1. BioLogic DuoFlow/Maximizer 10ml 91
4.2.2. BioLogic DuoFlow Maximizer 80 System 92
4.2.3. Column 1.1 cm x 22 cm packed bed (Steps 1 and 2) 92
4.2.4. Column 3.2 cm x 22 cm packed bed (Step 3) 93
4.3. Tip 94
5. Step 1 Purification Protocol Screening by Linear Salt Gradient 94
5.1. Overview 94
5.2. Duration 94
6. Step 2 Purification Protocol using a Step Gradient 96
6.1. Overview 96
6.2. Duration 96
7. Step 3 Purification Protocol using a Step Gradient and Simplified Analytics 98
7.1. Overview 98
7.2. Duration 98
8. Step 4 Offline pH Measurement and Calcium ion Analysis 99
8.1. Overview 99
8.2. Duration 99
9. Step 5SEC Profile for the Collected mAb Fraction and Regeneration Fraction 100
9.1. Overview 100
9.2. Duration 100
References 102
Referenced Literature 102
Chapter Seven: Salting out of Proteins Using Ammonium Sulfate Precipitation 104
1. Theory 105
2. Equipment 106
3. Materials 107
3.1. Solutions and buffers 107
4. Protocol 107
4.1. Preparation 107
4.2. Duration 107
5. Step 1 Removal of Proteins Marginally Soluble in (NH4)2SO4 108
5.1. Overview 108
5.2. Duration 108
5.3. Tip 108
6. Step 2 Precipitation of the Protein of Interest 108
6.1. Overview 108
6.2. Duration 111
6.3. Tip 112
6.4. Tip 112
6.5. Tip 112
6.6. Tip 112
6.7. Tip 112
References 113
Referenced Literature 113
Referenced Protocols in Methods Navigator 113
Chapter Eight: Using Ion Exchange Chromatography to Purify a Recombinantly Expressed Protein 114
1. Theory 115
2. Equipment 117
3. Materials 117
3.1. Solutions and buffers 117
4. Protocol 118
4.1. Duration 118
4.2. Preparation 118
5. Step 1 Equilibration of the Column 118
5.1. Overview 118
5.2. Duration 118
5.3. Tip 119
5.4. Tip 119
5.5. Tip 119
6. Step 2 Binding of the Protein Sample 120
6.1. Overview 120
6.2. Duration 120
6.3. Tip 120
6.4. Tip 120
7. Step 3 Removal of Unbound Proteins 120
7.1. Overview 120
7.2. Duration 120
8. Step 4 Elution of the Bound Protein 121
8.1. Overview 121
8.2. Duration 121
8.3. Tip 122
8.4. Tip 122
8.5. Tip 122
References 122
Referenced Literature 122
Referenced Protocols in Methods Navigator 122
Chapter Nine: Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins 124
1. Theory 125
2. Equipment 127
3. Materials 127
3.1. Solutions and buffers 128
4. Protocol 128
4.1. Preparation 128
4.2. Duration 129
5. Step 1 Standardization of the Gel Filtration Column 129
5.1. Overview 129
5.2. Duration 129
5.3. Tip 130
5.4. Tip 130
5.5. Tip 130
5.6. Tip 130
5.7. Tip 130
6. Step 2 Determination of the Sizes of Protein Species in a Sample 131
6.1. Overview 131
6.2. Duration 131
6.3. Tip 132
6.4. Tip 132
6.5. Tip 132
6.6. Tip 132
6.7. Tip 132
References 133
Referenced Literature 133
Referenced Protocols in Methods Navigator 133
Section IV: Protein Protocols/Purification of Membrane Proteins 134
Chapter Ten: Expression and Purification of Membrane Proteins 136
1. Theory 137
2. Equipment 140
3. Materials 140
3.1. Solutions and buffers 141
4. Protocol 143
4.1. Preparation 143
4.2. Duration 143
5. Step 1 Transformation of E. coli 145
5.1. Overview 145
5.2. Duration 145
5.3. Tip 145
6. Step 2 Cultivation of E. coli - Screening for the Optimal Expression Conditions 145
6.1. Overview 145
6.2. Duration 146
6.3. Tip 148
6.4. Tip 148
6.5. Tip 148
6.6. Tip 148
6.7. Tip 148
7. Step 3 Scale-Up Expression of a Membrane Protein Using the Optimal Expression Conditions 149
7.1. Overview 149
7.2. Duration 149
7.3. Tip 150
8. Step 4 Screening Detergents to Determine Optimal Solubilization of Membrane Protein 150
8.1. Overview 150
8.2. Duration 151
9. Step 5 Scale-up the Solubilization of the Membrane Protein 153
9.1. Overview 153
9.2. Duration 153
9.3. Tip 154
9.4. Tip 154
10. Step 6 Purification of Membrane Proteins Using Ni-NTA Superflow 155
10.1. Overview 155
10.2. Duration 155
10.3. Tip 157
References 159
Referenced Literature 159
Related Literature 159
Referenced Protocols in Methods Navigator 159
Chapter Eleven: Explanatory Chapter: Choosing the Right Detergent 160
1. Theory 161
2. Equipment 161
3. Materials 161
4. Protocol 163
4.1. Preparation 163
4.2. Tip 164
4.3. Caution 164
5. Using Detergents with Polyacrylamide Gel Electrophoresis 164
5.1. Tip 165
6. Using Detergents in Chromatograpy 165
6.1. Tip 165
6.2. Tip 165
7. Using Detergents with Optical Spectroscopy Techniques 166
7.1. Tip 166
8. Using Detergents with Mass Spectrometry Techniques 166
9. Using Detergents with Nuclear Magnetic Resonance (NMR) 167
10. Using Detergents in Protein Crystallization 167
10.1. Tip 167
References 167
Referenced Literature 167
Section V: Protein Protocols/SDS PAGE 168
Chapter Twelve: One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) 170
1. Theory 171
2. Equipment 171
3. Materials 172
3.1. Solutions and buffers 172
4. Protocol 173
4.1. Duration 173
4.2. Preparation 173
5. Step 1 Casting an SDS-PAGE Gel: Resolving Gel 173
5.1. Overview 173
5.2. Duration 173
5.3. Tip 174
5.4. Tip 174
5.5. Caution 174
6. Step 2 Casting an SDS-PAGE Gel: Stacking Gel 174
6.1. Overview 174
6.2. Duration 175
7. Step 3 Running an SDS-PAGE Gel 176
7.1. Overview 176
7.2. Duration 176
7.3. Tip 178
References 178
Referenced Literature 178
Referenced Protocols in Methods Navigator 178
Chapter Thirteen: Coomassie Blue Staining 180
1. Theory 181
2. Equipment 181
3. Materials 181
3.1. Solutions and buffers 181
4. Protocol 182
4.1. Preparation 182
4.2. Duration 182
4.3. Tip 182
4.4. Caution 182
5. Step 1 Stain a Gel Using Coomassie Blue 182
5.1. Overview 182
5.2. Duration 183
5.3. Tip 183
5.4. Tip 183
5.5. Tip 183
5.6. Caution 183
6. Step 2 Destain the Gel to Reduce Background Staining 184
6.1. Overview 184
6.2. Duration 184
6.3. Tip 185
6.4. Tip 185
6.5. Caution 185
Source References 186
Referenced Protocols in Methods Navigator 186
Chapter Fourteen: Silver Staining of SDS-polyacrylamide Gel 188
1. Theory 189
2. Equipment 189
3. Materials 189
3.1. Solutions and buffers 189
4. Protocol 191
4.1. Duration 191
4.2. Preparation 191
4.3. Tip 191
4.4. Tip 191
4.5. Tip 191
4.6. Tip 191
5. Step 1 Fix the Gel 191
5.1. Overview 191
5.2. Duration 191
5.3. Tip 192
6. Step 2 Stain the Gel 192
6.1. Overview 192
6.2. Duration 192
7. Step 3 Preserve the Gel 194
7.1. Overview 194
7.2. Duration 194
References 195
Referenced Protocols in Methods Navigator 195
Section VI: Protein Protocols/Standard in vitro Assays for Protein-Nucleic Acid Interactions 196
Chapter Fifteen: Standard In Vitro Assays for Protein-Nucleic Acid Interactions - Gel Shift Assays for RNA and DNA Binding 198
1. Theory 199
2. Equipment 203
3. Materials 203
3.1. Solutions and buffers 204
4. Protocol 205
4.1. Preparation 205
4.2. Duration 205
4.3. Caution 205
5. Step 1 Radiolabeling the Nucleic Acid Probe 206
5.1. Overview 206
5.2. Duration 206
5.3. Tip 207
5.4. Tip 207
6. Step 2 Bind Protein and Nucleic Acid 207
6.1. Overview 207
6.2. Duration 207
6.3. Tip 208
6.4. Tip 209
6.5. Tip 209
7. Step 3 Preparation of Polyacrylamide Gel 209
7.1. Overview 209
7.2. Duration 209
7.3. Tip 210
7.4. Tip 210
7.5. Tip 210
7.6. Tip 210
8. Step 4 Loading and Running Gel 210
8.1. Overview 210
8.2. Duration 211
8.3. Tip 211
8.4. Tip 211
9. Step 5 Analysis of Gel 211
9.1. Overview 211
9.2. Duration 212
9.3. Tip 212
9.4. Tip 213
9.5. Tip 213
9.6. Tip 213
9.7. Tip 213
9.8. Tip 213
References 214
Referenced Literature 214
Source References 215
Referenced Protocols in Methods Navigator 215
Chapter Sixteen: Protein Filter Binding 216
1. Theory 217
2. Equipment 217
3. Materials 217
3.1. Solutions and buffers 218
4. Protocol 218
4.1. Preparation 218
4.2. Duration 219
4.3. Tip 219
4.4. Caution 219
5. Step 1 Assemble Binding Reactions 220
5.1. Overview 220
5.2. Duration 220
5.3. Tip 220
5.4. Tip 220
5.5. Tip 220
5.6. Tip 220
5.7. Tip 221
6. Step 2 Quantify Binding 221
6.1. Overview 221
6.2. Duration 221
6.3. Tip 222
6.4. Tip 222
6.5. Tip 222
7. Step 3 Process Binding Data 223
7.1. Overview 223
7.2. Duration 223
References 224
Referenced Literature 224
Source References 224
Section VII: Protein Protocols/Troubleshooting Protein Expression 226
Chapter Seventeen: Explanatory Chapter: Troubleshooting Recombinant Protein Expression: General 228
1. Theory 229
2. Equipment 232
3. Materials 232
3.1. Solutions and buffers 233
4. Protocol 233
4.1. Preparation 233
4.2. Duration 233
5. Step 1 Monitoring E. coli cell Growth Before Induction 233
5.1. Overview 233
5.2. Duration 234
5.3. Tip 234
5.4. Tip 234
5.5. Tip 235
6. Step 2 Induction of Expression 235
6.1. Overview 235
6.2. Duration 235
7. Step 3 Monitoring E. coli Cell Growth After Induction 236
7.1. Overview 236
7.2. Duration 236
7.3. Tip 237
7.4. Tip 237
8. Step 4 Measurement of Protein Production 237
8.1. Overview 237
8.2. Duration 237
8.3. Tip 237
8.4. Tip 238
9. Step 5 Further Troubleshooting in E. coli 238
9.1. Overview 238
9.2. Duration 238
10. Step 6 Eukaryotic Expression Systems 242
10.1. Overview 242
10.2. Duration 244
References 246
Referenced Literature 246
Source References 248
Referenced Protocols in Methods Navigator 248
Chapter Eighteen: Explanatory Chapter: Troubleshooting Protein Expression: What to do When the Protein is not Soluble 250
1. Theory 251
2. Equipment 251
3. Materials 252
3.1. Solutions and buffers 252
4. Protocol 253
4.1. Preparation 253
4.2. Duration 253
5. Step 1 Centrifuge the Culture 254
5.1. Overview 254
5.2. Duration 254
5.3. Tip 254
6. Step 2 Lyse the Cells 254
6.1. Overview 254
6.2. Duration 254
6.3. Tip 255
6.4. Tip 255
6.5. Tip 255
6.6. Tip 255
6.7. Tip 255
6.8. Tip 255
7. Step 3 Remove the Cell Debris 256
7.1. Overview 256
7.2. Duration 256
8. Step 4 Analyze Protein Expression by SDS-PAGE 256
8.1. Overview 256
8.2. Duration 257
8.3. Tip 257
8.4. Tip 257
8.5. Tip 257
9. Step 5 Troubleshooting the Lack of Soluble Protein Expressed 258
9.1. Overview 258
9.2. Duration 258
References 263
Referenced Literature 263
Referenced Protocols in Methods Navigator 266
Section VIII: Protein Protocols/Western Blotting 268
Chapter Nineteen: Western Blotting using Chemiluminescent Substrates 270
1. Theory 271
2. Equipment 271
3. Materials 271
3.1. Solutions and buffers 272
4. Protocol 272
4.1. Duration 272
4.2. Preparation 272
5. Step 1: Protein Transfer to a Membrane 273
5.1. Overview 273
5.2. Duration 273
5.3. Tip 274
5.4. Tip 275
6. Step 2: Western Blot Detection using a Chemiluminescent Substrate 275
6.1. Overview 275
6.2. Duration 275
6.3. Tip 276
6.4. Tip 277
6.5. Tip 277
References 277
Referenced Literature 277
Source References 278
Referenced Protocols in Methods Navigator 278
Author Index 280
Subject Index 286

Chapter One

TCA Precipitation

Laura Koontz1, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, USA, 1Corresponding author: laurakoontz@gmail.com

Abstract

Trichloroacetic acid (TCA) precipitation of proteins is commonly used to concentrate protein samples or remove contaminants, including salts and detergents, prior to downstream applications such as SDS-PAGE or 2D-gels. TCA precipitation denatures the protein, so it should not be used if the protein must remain in its folded state (e.g., if you want to measure a biochemical activity of the protein).

Keywords

Bromophenol blue; Deoxycholate-trichloroacetic acid precipitation; Dithiothreitol (DTT); Glycerol; Sodium dodecyl sulfate (SDS); Sodium hydroxide (NaOH); Trichloroacetic acid (TCA); Tris base

1 Theory

TCA is an analog of acetic acid in which the three hydrogen atoms of the methyl carbon have been replaced with chlorine atoms. TCA is a relatively weak acid so it cannot hydrolyze the peptide bonds of proteins, but it does maintain an acidic pH in water. Addition of TCA to proteins in an aqueous solution disrupts the hydrogen-bonded water molecules (hydration sphere) surrounding a protein. These protein molecules no longer remain soluble and can be recovered by centrifugation. However, because TCA disrupts hydrogen bonding, the proteins will also lose their secondary structure and become denatured.

TCA precipitation has several drawbacks: (1) TCA precipitation denatures proteins and cannot be used if the activity of the protein is to be retained; (2) the protein concentration of the starting solution must be fairly high (≥ 5 μg ml− 1); (3) TCA precipitation does not work for all proteins and can be somewhat finicky. If TCA precipitation does not work for your protein of interest, it is advisable to try variants of TCA precipitation (included in this protocol) or other methods of precipitation or concentration, such as acetone precipitation, salting out (see Salting out of proteins using ammonium sulfate precipitation), immunoprecipitation (you can extract a protocol from Analysis of Protein-Protein Interactions by Co-immunoprecipitation), or ultrafiltration.

2 Equipment

Microcentrifuge

Vortex mixer

Magnetic stir plate

Aspirator

Micropipettors

Micropipettor tips

Ice bucket

Magnetic stir bars

1.5-ml microcentrifuge tubes

3 Materials

Trichloroacetic acid (TCA)

Sodium hydroxide (NaOH)

Deoxycholate (DOC, optional)

Tris base

Dithiothreitol (DTT)

Sodium dodecyl sulfate (SDS)

Bromophenol blue

Glycerol

Acetone (optional)

3.1 Solutions & buffers

100% TCA

Dissolve 100 g of TCA in 85 ml deionized water. Bring the volume up to 100 ml after all the TCA is in solution

Caution:

TCA can cause chemical burns and is harmful if inhaled

0.1 N NaOH

Dissolve 400 mg NaOH in 100 ml deionized water

Caution:

NaOH can cause chemical burns. Wear gloves and a lab coat

0.15% DOC

Dissolve 150 mg DOC in 85 ml deionized water. Bring the volume up to 100 ml after the DOC is completely in solution

10% SDS

Dissolve 10 g SDS in 85 ml deionized water. Bring the volume up to 100 ml after the SDS is completely in solution

10% SDS

Caution:

SDS and DOC are eye and lung irritants. Wear protective goggles and facemask

1 M Tris–HCl, pH 6.8 or pH 8.0

Add 121.14 g Tris base to 900 ml of deionized water. Adjust to final pH by adding 10 M or 1 M NaOH, dropwise. Adjust the volume to 1 l with deionized water

2× SDS loading buffer

Tris–HCl, pH 6.8

100 mM

1 M

5 ml

DTT

200 mM

1.5425 g

SDS

10%

20 ml

Bromophenol blue

0.2%

< 0.1 g

Glycerol

20%

100%

10 ml

Water

15 ml

Mix in a beaker with a stir bar until homogenous. Dispense into 1.5-ml microcentrifuge tubes as 1 ml aliquots

4 Protocol

4.1 Preparation

Prepare any of the chemicals and buffers listed above, if necessary.

Prepare your protein solution to be concentrated.

4.2 Duration

Preparation

About 2 h

Protocol

1 h to 1 day

4.3 Caution

Many of the chemicals used in the following protocols are harmful. Gloves and a lab coat should be worn and caution should be taken with all chemicals.

5 Step 1A Trichloroacetic Acid Precipitation

5.1 Overview

Addition of TCA will concentrate a protein and result in its denaturation. The protein concentration must be at least 5 μg ml− 1 to use this method.

5.2 Duration

45 min

1A.1 Add 100 μl of 100% TCA to 1 ml of the protein sample. Vortex the sample.

1A.2 Allow the protein to precipitate on ice for 30 min.

1A.3 Centrifuge the samples at 10 000× g at 4 °C for 5–15 min.

1A.4 Quickly and carefully aspirate the supernatant, taking care not to disrupt the pellet. The pellet should be whitish and fluffy.

1A.5 (Optional) Wash the pellet several times with 500 μl ice-cold acetone to remove any residual TCA. Centrifuge at 10 000× g at 4 °C for an additional 5 min afterward and aspirate the supernatant. Allow the pellet to dry.

1A.6 Resuspend the sample in 50 μl of 2× SDS buffer. Heat the sample at 100 °C for 5 min and analyze by SDS-PAGE (see One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)).

5.3 Tip

Keep all the reagents at 4 °C!

5.4 Tip

Precipitation can be carried out for 15 min at − 20 °C.

5.5 Tip

Bromophenol blue is a pH indicator, yellow below pH 4 and blue above that pH. Therefore, if the SDS sample buffer turns yellow, the solution is still slightly acidic due to remaining TCA. Just add a small amount of 1 M Tris pH 8.0, until the color returns to the usual blue (do not use NaOH because you could hydrolyze the protein!).

See Fig. 1.1 for the flowchart of Step 1A.

Figure 1.1 Flowchart of Step 1A.

6 Step 1B Deoxycholate-Trichloroacetic Acid Precipitation

6.1 Overview

The addition of deoxycholate (DOC) to a regular TCA precipitation will aid in precipitating proteins less concentrated than 1 μg ml− 1.

6.2 Duration

45 min

1B.1 Add 100 μl of 0.15% DOC to 1 ml of the protein sample. Vortex the sample.

1B.2 Incubate at room temperature for 10 min.

1B.3 Add 50 μl of 100% TCA and vortex the sample.

1B.4 Allow the protein to precipitate on ice for 30 min.

1B.5 Centrifuge the samples at 10 000× g at 4 °C for 5–15 min.

1B.6 Quickly and carefully aspirate the supernatant, taking care not to disrupt the pellet. The pellet should be whitish and fluffy.

1B.7 (Optional) Wash the pellet several times in 500 μl ice-cold acetone to remove any left over TCA. Centrifuge at 10 000× g at 4 °C for an additional 5 min afterward and aspirate the supernatant. Allow the pellet to dry.

1B.8 Resuspend the sample in 50 μl of 2× SDS buffer. Heat the sample at 100 °C for 5 min and analyze by SDS-PAGE (see One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)).

6.3 Tip

Keep all the reagents at 4 °C!

6.4 Tip

Precipitation can be carried out for 15 min at − 20 °C.

6.5 Tip

Erscheint lt. Verlag	22.3.2014
Sprache	englisch
Themenwelt	Medizin / Pharmazie
	Naturwissenschaften ► Biologie ► Biochemie
	Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
	Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
ISBN-10	0-12-420178-4 / 0124201784
ISBN-13	978-0-12-420178-1 / 9780124201781

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