Western blotting is likely the most used protein-analytical technique [1]. Labome surveys the literature citing Western blotting to understand the common usage of reagents and instruments involved.

Chemiluminescence is the commonly used approach for signal detection in Western blotting. Table 1 lists the major suppliers and their leading brands, and the number of citations among the formal publications surveyed by Labome. GE Healthcare with its various Amersham ECL kits and Thermo Fisher Pierce SuperSignal West kits dominate the provision of this Western blotting reagent. GE Healthcare provides three versions of ECL kits: regular ECL, Prime, and Select, based on sensitivity and signal lasting time. Figure 1 lists the selection guide from GE Healthcare. Fluorescence-based detection systems such as LI-COR IRDye or all-in-one flexible imaging systems like Bio-Rad ChemiDoc MP [2] are gaining popularity.

Dong JX et al used the Western Lightning Plus ECL substrate to detect HRP-conjugated secondary antibodies in Western blot [10]. Frottin F et al detected western blot signals through Luminata Forte Western HRP substrate from MilliporeSigma to study nucleolus [17]. Thermo Fisher SuperSignal West ECL reagents (mainly Femto and Pico) were used to study Cox-2 and mPGES-1 expression inmouse bone marrow–derived dendritic cells [18], the effect of increased potassium concentration in the tumor microenvironment on T cells [19], the role of the mGluR5-Erk pathway in tuberous sclerosis complex [20], the molecular mechanism of memory consolidation [21], and the mechanism for the suppressive effect of doxorubicin on tumor growth [22]. Kapogiannis D et al detected Western blot signals through LiCOR QuickWestern Kit (Cat# 926-68100) [13].

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Figure 1. Different versions of Amersham ECL kits provided by GE Healthcare. Courtesy of GE Healthcare.

The gels used in SDS-PAGE can be made freshly from acrylamide and other chemicals, or pre-cast gels from commercial suppliers can be used. Life Tech and Bio-Rad are the two major suppliers for pre-cast gels (Table 2). Kapogiannis D et al separated exosome proteins on 4-12% NuPAGE Bis-Tris Mini gels (NPO322BOX) from Thermo Fisher [13]. Thermo Fisher NuPAGE Novex Bis-Tris precast gels (mostly 4-12%) were used to investigate the functional property of the CK2 kinase in Drosophila [26], among others [27, 28]. Thermo Fisher NuPAGE Novex 3-8% Tris-acetate gels were used to investigate the functional property of CK2 kinase in Drosophila [26] and the regulation of telomerase by Wnt/beta-catenin signaling [29]. Thermo Fisher NuPAGE 4-20% Tris/Glycine gel was used to perform western blot to investigate the structural composition of the caveolar coat complex [28]. Bio-Rad 4-15% SDS-PAGE gels (5671084) were used to study lacteal junctions [30].

Two types of membranes are used for the protein transfer during the Western blotting: polyvinylidene difluoride (PVDF) and nitrocellulose membranes. Table 3 lists the major suppliers for both types of membranes. MilliporeSigma is the primary provider of PVDF membranes. Its immobilon PVDF membranes, for example, have been used to investigate choroid plexus organoids [11], Cox-2 and mPGES-1 expression in mouse bone marrow–derived dendritic cells [18], NLRP1B inflammasome [14], the involvement of L1 retrotransposon during cellular senescence [35] and the importance of the regulation of matrimony levels to study the oocyte-to-embryo transition in Drosophila [36]. The number 1 provider of nitrocellulose membranes is GE Healthcare, with the brand Amersham Hybond or Whatman Protran [37]. Its various formats of membranes were used to study the role of TRAF4 in modulating tight junctions and promoting cell migration [38], pheromonal ligands [39], protein synthesis initiation for MHC class I peptides [40], and the regulatory effect of PD-1 on IgA selection and the composition of gut microflora [41]. Pandolfini L et al transferred A549 cell proteins to Amersham Hybond-C Extra nitrocellulose membrane from GE Healthcare (RPN203D) for immunoblotting [3].

The basic steps of Wetern blot includes separation of proteins through a polyacrylamide gel, transfer of proteins to a membrane, hybridization of primary antibodies, and signal detection. Numerous variations of Western blot protocols exist. For example, Walma C and Collins JW detailed a protocol using nanoliter volumes of primary antibodies [42].

• 1x RIPA Buffer: 50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100 or NP40.
• 1x PBS Buffer: 137 mM NaCl, 2.7 mM KCl, 2.7 mM Na₂HPO₄, 2.7 mM KH₂PO₄, pH 7.4
• BCA protein assay kit or Bradford protein assay kit
• 1.5 M Tris buffer (pH 8.8): 90.68 g Tris-HCl to ddH₂O, adjust pH to 8.8 using HCl and to final 500ml
• 1.0 M Tris buffer (pH 6.8): 60.58 g Tris-HCl to ddH₂O, adjust pH to 6.8 using HCl and to final 500ml
• 10% APS: 100 mg AP in 1 ml ddH₂O. prepared before use.
• 10% SDS: 10 g SDS in 100 ml ddH₂O.
• 1x Tris-Glycine running buffer: 25 mM Tris, 230 mM Glycine (pH 8.3), 0.1% SDS.
• 3x SDS protein loading buffer: 150 mM Tris (pH 6.8), 6% SDS, 30% glycerol, 30 mM EDTA and 0.2% Bromophenol Blue. The buffer should be either freshly prepared or prepared, aliquoted, and frozen for future use.
• 1x TTBS: 25 mM Tris(pH 7.5): 0.15 M NaCl, 0.05% Tween-20, 0.001% Thimerosal
• 1x Transfer Buffer: 3 g Tris, 14.4 g Glycine and 200 ml methanol, add ddH₂O to 1L

Samples can also be lyzed in some of commercial lysis buffers, such as Pierce IP lysis buffer (Thermo Fisher, 87787) [43], instead of RIPA buffer.

Adherent cells

1. remove the supernatant and wash with 1X PBS to remove residual media.
2. add prechilled 400 ul-1 ml 1X RIPA buffer/100 mm dish. incubate on ice for 5-10 min.
3. scrape the cells completely and transfer to prechilled 1.5 ml microtubes on ice.
4. (optional) homogenize or sonicate thoroughly.
5. centrifuge at 12,000 rpm at 4°C for 10-15 min and collect supernatant for use.
6. total protein should be stored at -20°C until needed.

Supernatant cells

1. (optional) remove 100 ul aliquot for cell counting.
2. transfer cells to prechilled 1.5 ml microtubes or 15 ml tubes and centrifuge at 2000 rpm at 4°C for 5 min.
3. remove the media and resuspend the pellet with 1 ml prechilled 1x PBS and transfer to 1.5 ml microtubes.
4. centrifuge at 2000 rpm at 4°C for 5 min.
5. resuspend cell pellet with 1 ml prechilled RIPA buffer/10⁷ cells
6. incubate the cell suspension on ice with shaking for 30 min. or homogenize/sonicate thoroughly.
7. centrifuge at 12000 rpm at 4°C for 10-15 min and collect supernatant for use.
8. total protein should be stored at -20°C until needed.

1. tissue preparation and quantification. chop them into small pieces.
2. add about 500-600 ul prechilled 1x RIPA buffer/100 mg tissue.
3. homogenize the tissue thoroughly.
4. centrifuge at 12000 rpm at 4°C for 15-20min.
5. collect the supernatant for use.
6. total protein should be stored at -20°C until needed.

Bradford assay and BCA assay. See Labome review article on protein quantitation.

6%-15% resolving gel is made with stacking gel (5%) on the top and a gel comb (10 or 15 wells).

* 30% acrylamide mix (acrylamide:Bis = 29:1)

1. Mix well 2X SDS protein loading buffer with protein samples in 1:1 ratio thoroughly.
2. Pre-heating the sample at 50-60°C before loading gel. Pre-stained molecular weight markers are used to monitor protein separation and protein transfer efficiency.
3. Run the gel in 1X Tris-glycine buffer at 60-120V for 1-3 hours.

Transfer proteins to PVDF or NC* membrane for antibody detection.

1. Pre-wet materials such as gel, Whatman paper and sponge in 1X transfer buffer.
2. PVDF membrane should be incubated in methanol for 10 sec. to 1 min and then moved to 1X transfer buffer.
3. Stack the materials as following: case (black side), sponge, Whatman paper, gel, membrane, Whatman paper, sponge, case (clear side).
4. Place in the transfer apparatus with the black side facing black.
5. Transfer in a cooling environment with cold 1X transfer buffer. Transfer current and time should be optimized according to the blotting system manufacturer’s recommendations.

* never incubate NC membrane with methanol.

1. Primary antibody diluted in 1X TBST+3% BSA at the recommended dilution or optimize the dilution according to the results.
2. Incubate overnight or longer at 4°C or for 4 hours at RT.
3. Recover the primary antibody and store at 4°C. Then wash the membrane 3X 5-10 min on a shaker at RT.
4. Dilute the secondary antibody in 1X TBST and incubate the membrane for 1 hour at RT or for 2-4 hours at 4°C on a shaker.
5. wash 3X 10 min in TBST on a shaker at RT.

An estimate of the quantity and the concentration of a protein in a lysate can be made if a purified form of the protein is available [45]. Both the lysate and the purified protein are serially diluted in a Western blot. The signals from the purified protein are used to construct a calibration curve and the amount of the protein in the lysate can be calculated. Yasuda S et al, for example, quantified RAD23B and RAD23A in HCT116 cells using this approach, assuming 200 pg total protein per cell and 1 pl cell volume [46].

Western blotting and detection can be conducted in an integrated instrument through nano-volume capillaries (ProteinSimple) [47]. The system required less sample, antibody and time to run than conventional Western blot assay and was found to be highly sensitive, reproducible and quantitative over a large dynamic range [48]. ProteinSimple Western and traditional western blot were compared for analyzing lipid-rich adipose tissue samples and were found to highly correlative [49]. Its suitability for detecting erythropoiesis-stimulating agents was examined and found to be suitable for anti-doping evaluation in sports events [50] and for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy [48]. de Morree A et al used Peggy Sue capillary western to compare the expression of Pax3 protein in muscle stem cells between wild-type and Pax3-KO mice and among the wild-type mice treated with various antisense vivo-morpholino oligonucleotides [51]. More recent applications are examplified in [52].

Handler, D. et al discuss the pros and cons of using western blot as a tool validating the observed quantitative protein differences between, for example, two cell types, through mass spectrometry [53]. Western blot can be used to validate proteomics data if specific and sufficiently sensitive antibodies are used to confirm biologically relevant findings on a separate set of samples (distinct from the test set of samples for mass spec experiments), with fully disclosed validation strategy [53]. Western blot data cannot be used to imply confidence for low abundance results from high abundance data [53].

Different SDS-PAGE systems offer the resolution of denatured proteins of widely varying mass. The original Laemmli Tris-Glycine SDS-PAGE systems are widely used and are useful for the qualitative detection of target proteins of mass about 10-200kDa [54]. Tris-Glycine SDS-PAGE is the most commonly used PAGE system and uses a discontinuous buffer system with a highly alkaline pH of 8.6-9.5. The high pH of these gels makes them shelf stable only a month or two. Bis-Tris SDS-PAGE separates medium-sized proteins (20-250kDa) as well, but this system uses chloride anions and Bis-Tris cations and operates at the lowest pH of the systems described, pH of 6.4-7.1. This system can be run with either MES or MOPS denaturing buffer, with MES best for smaller proteins and MOPS for medium sized. The lower pH of these gels offers a longer shelf life and stability than do the basic Tris-Glycine gels. Other SDS-PAGE systems are useful when an application requires separation of very small or quite large proteins. Tricine-Tris SDS-PAGE separates small proteins (1-20kDa). The tricine replaces glycine in the Tris-Glycine system buffer and dramatically improves the visualization of small proteins because the front of SDS micelles that typically obscure tiny proteins is separated. Tris-Acetate SDS-PAGE is useful for separating very large proteins (<500kDa) and for studying oligomerization of proteins [55]. This system uses acetate as the negative ion in the gel buffer, tricine as the negative ion in the running buffer, with Tris as the cation in both. This system is useful for peptide sequencing and mass spectrometry and operates at pH of 7-8.1. A Tris-Acetate system used with a gradient gel can separate both very large and small proteins, from 10-500 kDa [55].

Question: I am planning to perform a Western Blot for native protein in Arabidopsis autophagy proteins. Could you please let me know how can I perform western bot for both my interested protein and reference gene (Tubulin). Do I have to blot the membrane two different times for my X protein and after washing with Tubulin then after washing for 2ndary HRP-antibody? Additionally, I want to detect the expression of autophagy native 3/4 proteins, can I perform all in one membrane?

Answer: You should not try to detect all the proteins at the same time. You should strip off the primary and secondary antibodies before using a second primary antibody. That is, blot with a primary antibody against the protein of your interest and a secondary antibody, detect with ECL, and then strip off the primary and secondary antibodies with a stripping buffer, and then blot with tubulin antibody and its secondary antibody, and detect. PVDF membranes are generally better than nitrocellulose membranes in retaining the proteins during stripping. The supplier of PVDF membranes should provide you with information about stripping buffers (mild or harsh) and the protocol. You should be able to perform the blot/strip cycles for a few times (up to three times in general). Each time before blot, you can check the blot with Ponceau S staining to visualize the sample proteins if you are not satisfied. Be aware that there will be the loss of the sample proteins on the blot due to stripping. However, during HRP development, oxidation of methionine, cysteine, and cystines (for non-reducing gels) may occur and may affect the affinity and specificity of some antibodies.

• wrong secondary antibody used for detection. (make sure the host of primary antibody)
• low concentration of primary/secondary antibody. (Raise the concentration and tried again)
• primary antibody does not recognize the protein in the species being detected.(check the specificity of primary antibody)
• protein amount loaded on the gel is too little. (increase the sample amount)
• transfer efficiency is quite low. (revise the manipulation of transfer procedure. Make sure PVDF pre-incubated with methanol. Dry the PVDF after the transfer to ensure the bonding of proteins to the hydrophobic membrane)
• primary antibody has been used too many times. (use a newly diluted primary antibody)
• blocking time is too long or wash too many times. (reduce blocking time and wash times)
• the detection kit does not work. (use positive control and make sure detection kit works well)
• sodium azide may inhibit the secondary antibody. (avoid using sodium azide in dilution buffer)
• incubation time with primary antibody or secondary antibody is too short. (extend incubation time)
• SDS loading buffer may have expired. (use freshly prepared SDS loading buffer)
• SDS loading buffer and sample lysate did not mix well. (vortex vigorously)

• Blocking time is too short or wrong blocking buffer used (extend blocking time or replace non-fat milk with BSA or make fresh milk solution). BSA, non-fat skim milk, and occasionally whole serum from goats or rabbits, etc. are used to pre-incubate the membranes to saturate any non-specific binding space on the membranes. Once the nonspecific binding space is saturated, the primary antibody will only bind to the specific antigen. BSA has one protein, albumin. Milk contains hundreds of proteins, with casein as its major component, and thus can block all potential non-specific binding sites. Therefore, milk is preferable over BSA, and cheaper too. Most primary antibodies are unlikely to cross-react with either albumin or casein. If any area without proteins has a high background, it is likely that the blocking step is not working properly.
• High concentration of primary/secondary antibody. (Reduce the antibody concentration)
• Insufficient wash. (increase wash times )
• Incubation temperature is too high. (make sure primary antibody incubation at 4°C)
• Wrong membrane or membrane dried out for a while. (prevent the membrane from drying)
• High background at higher molecular weight might indicate that reducing reagent/SDS/sample heating is not done properly.

• Primary antibody or secondary antibody is too high. (reduce the antibody concentration)
• ECL solution contaminated. (use freshly made ECL solution)
• ECL solution after mixing has a very short lifespan. (change to an ECL solution with a longer lifespan)

Too high voltage used or the buffer temperature is too high during gel electrophoresis. (reduce voltage and gel electrophoresis in a cold environment).

The buffer is likely problematic. Make fresh running buffer.

• High concentration of primary/secondary antibody. (reduce the antibody concentration)
• Protein amount loaded on the gel is too high. (reduce the loading amount)

Uneven transfer. (make sure the membrane is on gel evenly without bubbles)

High concentration of primary/secondary antibody. (reduce the concentration of antibody)

Endogenous immunoglobulins, especially in tissue lysate [56]. This signal should persist if a control without the primary antibody is run. Pre-clearance of endogenous immunoglobulins with, for example, Protein G agarose, using specific secondary antibodies such as TruBlot, or additional treatments like heating can help reduce the nonspecific signal from endogenous immunoglobulins [56].

An insufficient equilibration time of the gel or contact between gel and membrane is uneven. (make sure the gel is ok and improve the transfer procedure)

Bubbles between gel and membrane. (make sure there is no bubble between gel and membrane)

• Antibody specificity is poor. (change the primary antibody)
• Some proteins may migrate quite differently from their theoretical weight.

Post-translational modifications such as poly(ADP-ribosyl) – PAR chains) which introduce negative charges to protein molecules can not be resolved by SDS-PAGE [57]. They can however be revealed through CTAB-PAGE separation [57]. CTAB, Cetrimonium bromide ((C₁₆H₃₃)N(CH₃)₃Br, cetyltrimethylammonium bromide, hexadecyltrimethylammonium bromide), is an amine based cationic quaternary surfactant.

Dr. Jennifer Walker contributed the section on SDS-PAGE systems in Nov 2018.