Table 3.

An overview of western blotting procedure, key issues and possible solutions to address these issues.

Steps in Western blotting	Major issues	Possible Solutions	References
Sample preparation a) Protein extraction and purification	Frequent freeze/thaw cycles of tissue samples. Time gap between tissue collection and its freezing. Use of inappropriate lysis buffer. Poor homogenization techniques. Erroneous sample preparation and proteolytic degradation.	Optimize lysis buffer (detergent, salt, EDTA concentrations, pH) to prevent proteolytic degradation and high yield of target proteins. A suitable homogenization method must be chosen that can efficiently release the intracellular contents of the cell. Homogenization technique should be optimized for isolation of target proteins. To avoid endogenous proteolytic degradation samples should be quickly harvested in ice cold buffer (neutral pH) and immediately frozen in liquid nitrogen and stored at −80 °C until use. Multiple freeze/thaw cycles of protein samples must be avoided.	[3,5–7,19,129]
b) Measurement of protein concentration	Incorrect protein quantification due to incompatibility of buffer components. Reducing agents such as DTT often interfere with protein quantification methods.	Use protein quantification methods that are compatible with concentration of reducing reagents used in the lysis buffer. Appropriate lysis buffer must be used as blanks and in the standard curves to account for interference by buffer components.	[3,19]
c) Sample loading amount	Protein of interest is not within its linear detection range. Detection of low abundant proteins. Detection of post-translational modified proteins.	The amount of protein loaded on gels should be optimized to ensure linear dynamic range of sample protein and loading control in the blot. Use of advanced automated techniques such as capillary electrophoresis based western blotting methods may be useful in detecting low abundant and post-translationally modified proteins.	[7,23,24,40]
Gel electrophoresis Polyacrylamide gel	SDS-PAGE gels are not properly casted. Proteins are not properly resolved on gels.	Proper sample separation on SDS gels depends on protein samples containing SDS, DTT or 2-mercaptoethanol. Samples must be denatured by heating prior to loading. Optimization of separation conditions for target proteins (percentage of acrylamide, bis-acrylamide ratio) as well as amount of protein loaded on the gel for detection must be optimal. Routine check of pH of buffers used for electrophoresis is useful.	[3,19]
Blotting Transfer conditions and membrane	Poor and insufficient protein transfer. Low transfer efficiency of high and low molecular weight protein. Use of low quality membrane for transfer process. High background staining. Inefficient binding of protein to membrane.	Low molecular weight proteins can be efficiently transferred with the use of high quality nitrocellulose and PVDF membrane. Transfer conditions for both high and low molecular weight proteins must be optimized. Background fluorescence can be optimized using different combinations of blocking solutions and antibody concentrations. Use fresh transfer buffer and pre-stained markers to ensure optimize transfer.	[19,26]
Blocking	High amount of non-specific proteins on membrane and high background.	Optimize blocking time and solutions. Increase the percentage of non-fat dry milk or BSA used for blocking and antibody solutions.	[7,19]
Labelling Primary antibody	Multiple bands recognized by antibody tor the target protein. No bands or taint bands. Lot-to-lot variability in antibody. Lack to proper antibody vacation.	Optimize primary antibody dilutions, incubation time and temperature, and number to washes in washing butter. To minimize background, and non-specificity reduce primary antibody concentration and the amount to total protein loaded on gel. Validate all antibodies unless previously validate with lot specific data. Use to monoclonal antibodies over polyclonal antibodies is advantageous tor higher specificity to target protein. Use to recombinant antibodies has many advantages including the ability to engineer the antigen binding site, the ability to rapidly produce many variants to a specific antigen. Use positive and negative controls.	[3,7,19]
Secondary antibody	Non-specific bands, high background, ghost bands.	Reduce secondary antibody concentration.	[3,7,19]
Detection and imaging	Saturate band signal. Signal too high or too low.	Use shorter exposure time. Optimize incubation time and amount to enhanced chemiluminescent reagent. Use digital imagers over film-based processors tor higher sensitivity, and better linearity. Use fluorescent probes tor western blot.	[7,19]
Quantification Densitometry analysis to target protein using afferent software	Inaccurate quantification to protein to interest, poor image quality, and signal saturation.	Optimize quantification using well established antibodies and sample lysates. Use two different software tor quantification. Use software that shows signal saturation.	[7,15,19,35,129]
Normalization of housekeeping protein	Loading control and the target protein band intensities are not within the same linear range. The expression to housekeeping proteins can sometimes vary with type to tissue and aitterent experimental conditions.	The linear dynamic range tor the total protein, the housekeeping protein ana the target protein needs to be determined and used within this range tor accurate western blots. Use ot a total protein stain as an alternate loading control (Ponceau S or Bio-Rad’s Stains-tree methods).	[15,19,37,38]

Abbreviations: polyvinylidene difluoride (PVUF); dithiothreitol (DTT); Sodium dodecyl sulphate (SUS); polyacrylamide gel electrophoresis (PAGE).