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Guide to Western Blot Sample Preparation

21 Aug 2019

Guide to Western Blot Sample Preparation

Western blotting (WB) is a widely used technique to detect protein expression in cells and tissues. The first step in the WB procedure is preparation of tissue or cell lysate. Choosing the proper buffers and running conditions are key steps in ensuring the success of the experiment.

 

Lysis Buffers

 

Lysis buffers vary from gentle, containing no detergents, to harsher denaturing solutions (i.e. RIPA) containing sodium dodecyl sulfate (SDS) and other ionic detergents. Choosing a lysis buffer depends on the sublocalization of the protein. Typically, mild non-ionic detergents such as NP-40 are used for extraction of soluble cytoplasmic proteins. Harsher buffers such as RIPA are used for isolation of membrane bound proteins and nuclear proteins. Table 1 and Table 2 provide lysis buffer suggestions based on the source of protein and commonly used lysis buffer recipes. Some proteins, such as histones, or tissue samples may require an additional sonication step to fully release the proteins. Sonication is also used to break down the DNA to reduce viscosity of the solution.

 

Table 1. Common Lysis buffers used to prepare samples for Western Blot
 
Buffer Protein Localization
Tris-HCl Cytoplasmic (Soluble)
Tris-Triton X-100 Cytoplasmic (Membrane Bound)
NP-40 Whole Cell Lysate or Membrane Bound
RIPA Nucleus, Mitochondria, Membrane Bound

 

Table 2. Compositions of the most common Lysis buffers used in Western Blot

Buffer Composition
Tris-HCl 20mM Tris-HCl, pH 7.5
Tris-Triton X-100 10 mM Tris, pH 7.4 
100 mM NaCl 
1 mM EDTA 
1 mM EGTA 
1% Triton X-100 
10% glycerol 
0.1% SDS 
0.5% deoxycholate
NP-40 150mM NaCl 
1% NP-40 
50mM Tris, pH 8.0
RIPA 150 mM NaCl
1.0% NP-40 or Triton X-100
0.5% sodium deoxycholate
0.1% SDS
50 mM Tris, pH 8.0

 

Protease and Phosphatase Inhibitors

 

Other important components of lysis buffers are protease and phosphatase inhibitors that prevent fast proteolysis and dephosphorylation of the targets upon cell lysis. To slow protein degradation we advise preparing the buffers fresh before each experiment, and keeping cells and buffers on ice during the whole procedure. Table 3 details the most common inhibitors used in Western blot. Most of these are now available as ready-to use cocktails:

 

Table 3. Functions of the most commonly used protease and phosphatase inhibitors

 

Inhibitor Target
Leupeptin Lysosomes
Pepstatin A Aspartic proteases
AEBSF Serine proteases
Aprotinin Trypsin, Chymotrypsin, Plasmin, Thrombin
Bestatin Amino peptidases
E-64 Cysteine proteases
PMSF Serine proteases
NaF Pyrophosphate 
β-glycerophosphate
Serine & Threonine phosphatases
Orthovanadate Tyrosine phosphotases
EDTA Mg2+ and Mn2+ metalloproteases

 

Determining protein concentration

 

Before loading protein on the gel it is important to determine the protein concentration and normalize it across the samples of each experiment. The protein concentration can be determined using absorbance at 280 nm, Bradford (Coomassie-based) or BCA assay. Presence of SDS in the lysis buffer can interfere with the Bradford assay. In this case, BCA assay must be used. We advise loading approximately 15-30 μg of the protein lysate to ensure a proper range of protein detection.

 

Protein loading

 

Before loading the samples, dilute them in a gel loading buffer, such as 2x Laemmli sample buffer. It contains reducing and denaturing agents including SDS, β-mercaptoethanol, and/or DTT. Glycerol allows protein to stay inside the well, and the dye bromophenol blue helps track the protein movement. 

 

Table 4. Composition of 2X Laemmli buffer

2x Laemmli buffer (pH6.8) Function
4% SDS Protein denaturation
5% β-mercaptoethanol or DTT Reduced disulfide bonds leading to protein unfolding
20% Glycerol Increases sample density for protein loading to 
the well
0.004% Bromophenol Blue Enables visualization of protein migration on the gel

 

Loading and running buffer conditions

 

For a routine Western blot, it is recommended to run the gel in reducing/denaturing conditions. For this, the lysate must be boiled in sample buffer at +95-100°C (5 minutes) or at +70°C (10 minutes). Then, samples can be immediately loaded on a gel or stored at -20°C for later analysis. 

In some circumstances, the target specific antibody is not able to recognize the denatured form of the protein. In this case, non-denaturing conditions can be achieved by avoiding the use of SDS. 

Certain antibodies only recognize proteins in their non-reduced or oxidized forms. In this case, the reducing agents such as β-mercaptoethanol (β-ME) or DTT should not be included in the buffer. Refer to Table 5 below describing the loading buffer and gel running buffer conditions for each purpose.

 

Table 5. Loading and Running buffer conditions

 

Condition Sample Loading Buffer Gel Running Buffer Comment
Reduced and Denatured SDS β-ME or DTT SDS Removes high order protein structure including disulfide bonds.
Reduced No SDS β-ME or DTT No SDS High order protein structure is present, but the disulfide bonds are removed.
Denatured SDS No β-ME or DTT SDS Removes high order protein structure, but the disulfide bonds are present.
Native No SDS No β-ME or DTT No SDS The protein is in its native form. Multimers and protein-protein interactions can be observed.

 

In the end, don't forget to load proper positive and negative controls, including:
  • Protein ladder to identify the bands of correct size.
  • Negative control - cell or tissue lysate that doesn't express the protein of interest.
  • Positive control - cell or tissue lysate that is known to express the protein of interest.
  • Run a separate gel with only secondary HRP conjugated antibodies to help rule out any non-specific bands.our 
Optimizing the lysis buffer and gel loading/running buffer is one of the first important steps in ensuring the success of your Western blot procedure. For more tips and tricks to help optimize your experiment, please check Western Blotting webpage and Technical Protocols.

 

Dr Ekaterina Zvezdova

BioLegend

21 Aug 2019

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