The first step in sample preparation is isolating target proteins from their source, cells or tissues via lysis. Choosing the appropriate lysis buffer is the first step of your WB to success. The two major considerations are whether the chosen antibody will recognize denatured samples and the expression location of your protein of interest. Lysis buffers differ in their ability to solubilize proteins, with those containing sodium dodecyl sulfate (SDS) and other ionic detergents considered to be the harshest and therefore most likely to give the highest yield.
|Cellular Location||Buffer Recommended|
|Nuclear / Mitochondria||RIPA, or nuclear /mitochondria fractionation for increased protein of interest concentration|
|Cytoplasmic (soluble)||Tris-HCl buffer|
|Cytoplasmic (cytoskeletal bound)||Tris-Triton buffer|
|Membrane-bound||NP-40, RIPA or Tris-Triton buffer|
Common Lysis Buffer Recipes
Protease and Phosphatase Inhibitors
Immediately following cell lysis, proteolysis, dephosphorylation, and denaturation begin to occur. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer. Ready-to-use cocktails of inhibitors from various suppliers are available.
|Inhibitor||Specificity of Inhibitor||Final Concentration in Lysis Buffer||Stock (store at -20°C)|
|Aprotinin||Trypsin, Chymotrypsin, Plasmin||2 µg/mL||Dilute in water, 10 mg/mL. Do not re-use thawed aliquots.|
|Leupeptin||Lysosomal||5-10 µg/mL||Dilute in water. Do not re-use thawed aliquots.|
|Pepstatin A||Aspartic Proteases||1 µg/mL||Dilute in methanol, 1 mM.|
|PMSF||Serine, Cysteine proteases||1 mM||Dilute in ethanol. You can re-use the same aliquot.|
|EDTA||Metalloproteases that require Mg2+ and Mn2+||5 mM||Dilute in dH2O, 0.5 M. Adjust pH to 8.0.|
|EGTA||Metalloproteases that require Ca2+||1 mM||Dilute in dH2O, 0.5 M. Adjust pH to 8.0|
|Sodium Fluoride||Serine/threonine phosphatases||5-10 mM||Dilute in water. Do not re-use once defrosted.|
|Sodium orthovanadate||Tyrosine phosphatases||1 mM||Dilute in water. Do not re-use once defrosted|
Preparation of Lysate from Cell Culture
Note: 1 mL/ 107 cells/100 mm dish/150 cm2 flask; 0.5 mL/ 5x106 cells/60 mm dish/75 cm2 flask.
Note: The centrifugation force and time depending on the cell type. A guideline is 20 min at 12,000 rpm.
Preparation of Lysate from Tissues
Note: Volumes of lysis buffer must be determined in relation to the amount of tissue present.
Determination of Protein Concentration
In order to ensure equal loading of each lane, determination of protein concentration is important. There are some commonly used protein assay methods to determine protein concentration in biochemical laboratories. According to your experiment conditions, you can perform one assay to determine protein concentration.
|Sample Composition||Protein Assay Not Recommended||Protein Assay Recommended|
|Arginine-rich protein||Bradford assay||Lowry, BCA or UV assay|
|Cysteine-rich protein||BCA assay||Lowry, UV or Bradford assay|
|Tryptophan- or tyrosine-rich (no extinction coefficient)||UV assay||Lowry, UV or Bradford assay|
Preparation of Samples for Loading into Gels
To denature, use a loading buffer with the anionic detergent SDS, and boil the mixture at 100°C for 5 min. The standard loading buffer is called 2X Laemmli buffer. The 2X is to be mixed in 1:1 ratio with the sample.
2X Laemmli Buffer Recipe
Check the pH and bring it to pH 6.8
An antibody may recognize an epitope made up of non-contiguous amino acids. Under these circumstances, it is important to run a WB in non-denaturing conditions. Generally, a non-denaturing condition simply means leaving SDS out of the sample and migration buffers and not heating the samples. Besides, the reducing agents β-mercaptoethanol and DTT must be left out of the loading buffer and migration buffer.
|Protein State||Gel Condition||Loading Buffer||Migration Buffer|
|Reduced, denatured||Reducing and denaturing||With β-mercaptoethanol or DTT and SDS||With SDS|
|Reduced, native||Reducing and native||With β-mercaptoethanol or DTT and SDS||No SDS|
|Oxidized, denatured||Non-reducing and denaturing||No β-mercaptoethanol or DTT, with SDS||With SDS|
|Oxidized, native||Non-reducing and native||No β-mercaptoethanol or DTT, with SDS||No SDS|
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