The first step in sample preparation is isolating proteins from their source. Usually, proteins are isolated from cells or tissues via lysis. Lysis breaks down the cell membrane to separate proteins from the non-soluble parts of the cell. A number of lysis buffers can be used to prepare samples for western blotting. In general, these buffers vary in the strength of their detergents to release soluble proteins. Stronger detergents, such as Triton X-100 are recommended for difficult to solubilize proteins. Refer to the list below for common lysis buffer recipes.
The sub-cellular localization of the protein of interest can be used as a starting point to determine the optimal lysis buffer to obtain high protein purity and yield. Proteins found predominantly or exclusively in a sub-cellular location, such as nucleus or mitochondria, can be enriched with fractionation kits. This is particularly useful when probing weakly expressed proteins. Since each protein is different, lysis buffer and detergent conditions may require optimization for individual western blotting experiments. Refer to the table below as a starting point to choose a lysis buffer.
Lysis buffer recommendations
Cellular Location
Recommended Buffer
Whole Cell Lysate
NP-40
Nucleus
RIPA
Mitochondria
RIPA
Cytoplasm
Tris-HCl
Membrane-bound Protein
RIPA (SDS is generally considered harsh and thus is often well-suited for difficult to solubilize proteins)
Common lysis buffer recipes
Buffer
Components
NP-40
150 mM NaCl
1% NP-40 or Triton X-100
50 mM Tris pH 8.0
RIPA
150 mM NaCl
1% NP-40 or Triton X-100
0.5% sodium deoxycholate
0.1% SDS
50 mM Tris, pH 8.0
Tris-HCl
20 mM Tris-Hcl, pH 7.5
Protease and phosphatase inhibitors
Immediately following cell lysis, proteolysis, dephosphorylation, and denaturation begin to occur. This activity should be kept to a minimum by preparing samples on ice or at 4˚C and by adding protease and phosphatase inhibitors fresh to the lysis buffer.
While there are many commercially available ready-to-use inhibitor cocktails (often proprietary), a homemade mix can be made based on individual needs. The table below lists common protease and phosphatase inhibitors, their targets, and the recommended final concentration in the lysis buffer.
Common protease and phosphatase inhibitors
Inhibitor
Target
Final Concentration
Aprotinin
Trypsin, chymotrypsin, plasmin
2 µg/mL
Leupeptin
Lysosomal
1-10 µg/mL
Pepstatin A
Aspartic proteases
1 µg/mL
PMSF
Serine proteases
1 mM
EDTA
Mg2+ and Mn2+ metalloproteases
1-5 mM
EGTA
Ca2+ metalloproteases
1 mM
Sodium fluoride
Serine & threonine phosphatases
5-10 mM
Orthovanadate
Tyrosine phosphatases
1 mM
Pyrophosphate
Serine & threonine phosphatases
1-2 mM
B-glycerophosphate
Serine & threonine phosphatases
1-2 mM
CARRYING OUT LYSIS
The cell lysis protocol can vary widely depending on the cell or tissue sample used. For example, lysing heart or brain tissue from a mouse may require homogenization, which typically involves flash freezing the sample in liquid nitrogen prior to grinding with a mortar and pestle or an electric homogenizer. A lysis protocol should be chosen based on standard protocols in the field for the given tissue. The following is a general example of a protocol for lysing cells grown in culture.
Lysate preparation from adherent cell culture
Wash cell culture dish on ice with ice-cold PBS.
Aspirate PBS and add ice-cold lysis buffer (1 mL per confluent 107 cells/100mm dish/150 cm2 flask). See the table above for lysis buffer recommendations based on the subcellular location of the protein of interest.
Using a cell scrapper, scrape adherent cells off the dish and transfer the cell suspension into a microcentrifuge tube. If required, the cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer.
Agitate cells for 30 minutes at 4˚C
Centrifuge cell lysate mixture at 4˚C.
Note: The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm.
Transfer the supernatant (lysate) to a fresh tube on ice.
DETERMINING LYSATE PROTEIN CONCENTRATION
It is important to determine the protein concentration within each lysate in order to ensure equal loading of the SDS-PAGE gel in subsequent steps. Equal loading allows proteins levels and expression differences to be accurately quantified in western blotting. Protein concentration can be determined by performing a standard Bradford, Lowry, or BCA assay. Protein samples can be frozen at -20 ˚C or -80 ˚C for later use or prepared for gel loading for immediate use.
PREPARING LYSATES FOR GEL LOADING
The epitope usually resides within the 3D conformation of the protein. Thus, it is necessary to unfold or denature the protein to enable access to the antibody. Denaturing is performed by briefly boiling the sample in a loading buffer containing SDS.
The most common loading buffer for SDS-PAGE gel electrophoresis is 2X Laemmli buffer. It can also be made at 4X or 6X concentration, which may be helpful if larger volumes of lysates with low protein concentration.
Loading buffer recipe:
Loading Buffer
Components
2X Laemmli buffer
4% SDS
5% 2-mercaptoethanol
20% glycerol
0.004% bromophenol blue
0.125 M Tris HCl
Adjust pH to 6.8
Understanding the loading buffer components:
Component
Notes
SDS
Causes proteins to become negatively charged by their attachment to SDS anions. The SDS wrapping around the polypeptide backbone causes protein denaturation. The negative charged conferred by SDS to polypeptide chains is proportional to their length. Because of this, proteins can be separated by SDS-PAGE electrophoresis according to their molecular weight and not by intrinsic electrical charge. Both the loading buffer and the gel running buffer contain SDS to allow this.
Reducing agents: β-ME or DTT
While SDS serves to unfold proteins, reducing agents further remove tertiary and quaternary structure by reducing intramolecular and intermolecular disulfide bonds, converting proteins to a linear form.
Glycerol
Is added to sample loading buffer to increase sample density so, when samples are loaded into the gel, they sink to the bottom of the well. This promotes even protein loading between wells and minimizing sample overflow.
Bromophenol blue
Bromophenol blue is a small anionic dye that is added to enable the visualization of protein migration during gel electrophoresis. Because of its small size, bromophenol blue migrates faster than the proteins in the samples and provides a migration front to monitor the electrophoresis process and prevent sample run-off.
Loading and running buffer conditions
Protein state
Sample loading buffer
Gel running buffer
Reduced and denatured (most common)
SDS + β-ME or DTT
SDS
Reduced and native
β-ME or DTT, No SDS
No SDS
Oxidized and denatured
SDS, No β-ME or DTT
SDS
Oxidized and native
No SDS and No β-ME or DTT
No SDS
Sample preparation for gel loading
Determine the protein concentration of each cell lysate.
Determine how much protein to load (Recommended: 10-50 μg/lane) and add an equal volume of 2X Laemmli buffer.
Reduce and denature the samples by boiling the lysates in Laemmli Buffer at 95-100˚C for 5 minutes.
Note: This step should only be skipped if the antibody datasheet recommends non-reducing or non-denaturing conditions. Occasionally, antibodies only recognize epitopes as they exist on the surface of a protein’s native, non-denatured state. Non-denaturing conditions can be generated by leaving SDS out of the sample and migration buffers and not boiling the samples. Further, certain antibodies only recognize proteins in their non-reduced, or oxidized forms. In this case, the reducing-agents β-mercaptoethanol (βME) or DTT should not be included in the buffers. Refer to the table above for loading buffer and gel running buffer guidelines.
