The primary species reactivity as well as the validating tissue used are listed on the data sheets for each antibody that can be found on the ASB web site. Secondary Species Reactivity: All secondary species reactivities were determined by computational sequence homology analysis, comparing the homology between the immunizing peptide sequence and the NCBI database for each specific species.
Using a small pair of broad-tipped forceps, place the coverslips carefully in the Antigen Retrieval Buffer in the coverglass staining jar, making note of which side of the coverslips the cells are on.
Optimal times should be determined by the researcher. Remove the coverslips from the Antigen Retrieval Buffer and allow them to come to room temperature. Immerse coverslips with the side containing the cells facing up, in PBS, in the 6-well tissue culture plates. Rinse the cells 3 times in PBS.
Continue with blocking and primary antibody staining steps. Deparaffinize sections in 2 changes of xylene, 5 minutes each. Rinse in distilled water.
This is particularly important for anti-goat conjugates see 4. Attention when using anti-goat conjugates. Anti-goat secondary antibodies from the host species donkey, rabbit, and mouse highly cross-react to bovine immunoglobulins, for example in milk powder. If the secondary antibody dilution buffer contains milk powder, almost all of the anti-goat conjugate will be adsorbed by bovine Ig. This leads to either a complete lack of signal or a faint signal, if the conjugate is applied at high concentrations.
Additionally these conjugates may produce background on the blot, if bovine milk powder was used for blocking. For the detection of goat primary antibodies on Western blots, we recommend anti-goat from bovine cat. Endogenous immunoglobulins in sample lysate. If the sample material separated by SDS-PAGE is a lysate of immunoglobulin-containing cells or tissues, secondary antibodies without cross-reaction MinX to the same species than the sample should be used.
Alternatively, normal serum Ig of the same species than the sample material can be added to the secondary antibody dilution buffer. Background bands from antibodies after immunoprecipitation. In the following article you can read how background bands caused by precipitating immunoglobulins on the blot can be avoided when using secondary antibodies for Western blot detection.
Immunoblot Western Blot — Avoiding background bands from antibodies. One of the most common problems in immunoblotting Western blot using secondary antibodies for indirect detection are background bands caused by antibodies in sample preparations originating from, e.
Under non-reducing conditions gamma immunoglobulins IgG show a band at approximately kDa. B, membrane was incubated with anti-MRP1 rabbit antibody ab and mouse anti-vinculin antibody ab at a dilution and , dilution, respectively. Separate images were required to visualize HiMark TM pre-stained protein standard. White dotted line and scissor symbol denote splicing.
The host cell DNA repair machinery may then disrupt the genetic locus through a frameshift mutation. These KO samples provide valuable tools for antibody validation, as a loss of signal during testing confirms that the tested antibody has bound to its intended target.
Small or short interfering RNAs siRNAs can induce transient silencing of the target gene and suppress target protein expression On a Western blotting, siRNA KD can significantly decrease, or even completely suppress, the intensity of the target protein band, compared with the untreated sample. The extent of KD or KO must be validated experimentally before the sample is used as a control for antibody validation.
This validation should include both genomic and proteomic approaches. Although pre-adsorption tests are not recommended for antibody validation, some researchers do use pre-adsorption controls in immunohistochemistry and other immunoassays 8 , 14 , 17 , — 19 , 39 , If antibody binding is specific to its immunizing peptide, pre-adsorption will substantially decrease the intensity of protein staining.
This method has several limitations, however. Although this test may prove that an antibody is specific, it cannot validate that it is selective 18 , Pre-adsorption controls may be most useful when using crude sera As a monoclonal or affinity-purified polyclonal antibody has already been selected for its ability to bind the target antigen, pre-adsorption provides little additional information.
Overall, routine use of pre-adsorption controls for antibody validation is discouraged 16 , 18 , 41 , Orthogonal validation requires transcriptomics or antibody-independent proteomics to validate the differential protein expression seen with antibody assays. The transcriptomic analysis incorporates mRNA-based assays 43 , 44 and gene-expression assays, such as luciferase reporters 45 , 46 , to determine whether sample gene expression correlates with antibody-binding patterns.
Although these strategies can be used where endogenous protein expression is not well-characterized, they can be compromised by a lack of correlation between RNA expression and protein abundance 86 , along with minimal variation in sample protein expression Importantly, transcriptomic analysis will struggle to conclusively validate antibodies to post-translational modifications. This is because post-translational processing is often determined by cell signaling and protein interactions rather than gene expression.
Proteomic orthogonal validation includes targeted proteomics and MS 48 , Although these are valuable tools for confirming antibody specificity through an analysis of protein levels between samples, they have issues differentiating co-migrating proteins of a similar molecular weight However, these tools are most effective when combined with antibody assays, such as immunoprecipitation MS that can identify cross-reactive antigens through peptide mass fingerprinting Other independent validation approaches may include ELISA, immunohistochemistry, immunocytochemistry, tissue microarrays, and peptide arrays or reverse-phase protein arrays 5 , As discussed previously, an antibody used for Western blotting is not necessarily suitable for use in other applications.
An independent antibody-based assay may therefore use a different antibody to the one undergoing Western blotting validation. Confirmation of Western blotting results via independent assays may provide biological verification or validation of an observed experimental effect.
Importantly, findings of independent experimental approaches help to determine whether a finding is biologically relevant and is potentially applicable to other samples or experimental systems. It is important to consider the state of the proteins within the independent assay and the impact this may have on antigen recognition by the antibody. Formalin-fixed, paraffin-embedded tissue samples in immunohistochemistry may present a radically different range of epitopes when compared with denatured and reduced protein immobilized onto a membrane.
Therefore, evidence of specificity within an independent assay can support, but not substitute, evidence of specificity within Western blotting experiments. In a Western blotting experiment, the ratio of the target to other proteins in the sample is skewed, and unrelated sample proteins are typically present in considerable excess. The antibody must be able to locate and selectively bind the target antigen in a complex mixture. Target protein concentration strongly influences antibody selectivity, and therefore purified or overexpressed target protein should not be used as the sole protein sample for antibody validation.
Overexpression of the target alters the balance of protein abundance in assays, giving the antibody an artificial advantage 5 , 54 , An overexpressed target may mask off-target binding that creates false-positive results at endogenous target expression levels. If an affinity-tagged protein is used for validation it should be expressed at endogenous levels 5. Purified protein gives no opportunity to evaluate off-target binding but can be valuable for confirming antibody reactivity with the target.
Nevertheless, endogenous samples should always be present to evaluate potential off-target binding Fig. Validation of IDH1 antibody using purified recombinant protein in multicolor and chemiluminescent Western blotting. Some overspill of the recombinant protein into neighboring lanes is observed white box. C, when both and nm channels are displayed, the signal from ab32 and ab overlaps at 50 kDa, identifying the c-Myc—tagged IDH1 protein. Chameleon TM Duo pre-stained protein ladder for accurate sizing of protein bands.
D, single blot was split into two halves green line to be incubated with either rabbit anti-IDH1 antibody ab; 0. Antibody concentration and incubation time can affect selectivity and nonspecific binding 11 , A high-affinity primary antibody can bind and detect its antigen at a low working concentration, whereas a low-affinity antibody will require a higher working concentration.
By contrast, higher working concentrations increase the likelihood of off-target binding and cross-reactive bands that could mask the protein of interest. The optimal concentration of the primary antibody that produces the best blot signal—to—noise ratio should be determined experimentally. A product's recommended usage conditions can be taken as guidelines; however, they may be based on different assay conditions or reagents. A titration experiment with a series of antibody dilutions is a practical way to do this Table 3 has guidelines on the dilution of unpurified antibodies.
Unpurified antibodies will not have a concentration stated on the vendor datasheet. For most whole antisera, culture supernatants, or ascites fluid products, the concentration may be unknown. Unpurified antibody preparations vary significantly in specific antibody concentrations. If the specific antibody concentration of an unpurified antibody preparation is unknown, these concentration estimates may be used as a rough guideline.
Please remember that these dilutions and concentration estimates are only a starting point, and dilutions may need to be adjusted based on the experimental results.
NA, not applicable. For the best results, it is good practice to maintain consistent experimental conditions by choosing a fixed incubation time with a consistent secondary antibody concentration and to test each primary antibody dilution on the same type of sample. Secondary antibody concentration also affects the selectivity of a primary antibody. Too much secondary antibody will increase off-target binding on a blot, and too little may produce faint signals.
Note that the recommended dilution range depends on the type of secondary antibody conjugate and detection method selected. For example, the recommended dilution for an HRP conjugate and chemiluminescent detection may not be appropriate for a fluorescent antibody conjugate. Monoclonal antibodies have similar batch—to—batch consistency, and it may only be necessary to perform the titration once.
However, especially for polyclonal antibodies, there can be differences between batches of the same antibody, and it is good practice to perform a titration for each new batch. A blocking buffer should reduce the nonspecific binding of antibodies to off-target sample proteins on the membrane and to the membrane itself. It should also stabilize the blotted sample proteins without disrupting their retention on the membrane surface 57 , — This enhances the specificity and selectivity of the antibody, increasing the signal—to—noise ratio for detection of the target.
Blocking buffers should not mask the interaction of the antibody with the target antigen or display enzymatic activity or other properties that may interfere with antibody—antigen interaction or target protein detection Bovine serum albumin BSA , nonfat dry milk, and casein are commonly-used blocking buffers that differ in blocking strength and may interfere with target protein detection in some assay contexts.
Nonfat dry milk is inexpensive and widely used. It has a high blocking strength that may disguise some antigens and can also inhibit biotin—streptavidin interactions Endogenous biotin and IgGs in milk can also potentially cross-react with sheep or goat secondary antibodies.
Casein, an abundant protein in found in nonfat dry milk, is a phosphoprotein that may cross-react with some phospho-specific antibodies and increase background signal BSA is a popular alternative to milk-based blockers and may be more suitable for detection of antibodies labeled with biotin or alkaline phosphatase 57 , However, some sources of BSA contain phosphotyrosine residues that may bind anti-phosphotyrosine antibodies, and endogenous carbohydrates may interfere with detection of lectins 57 , BSA blockers can increase nonspecific banding.
Nonmammalian blocking agents are less likely to contain cross-reactive epitopes and may produce lower background signal than mammalian reagents, which are often available from suppliers of fluorescent Western blotting platforms.
Excessive blocking can cause loss of sample proteins from the surface of the membrane, particularly with nonfat dry milk. Den Hollander and Befus 63 demonstrated nonselective elution of blotted proteins from the membrane in proportion to the amount of milk used, with progressive loss over time. The strength of blocking agent and length of incubation should be considered when staining for low abundance proteins. Membrane blocking, antibody incubations, and washes often use Tris-buffered saline TBS or phosphate-buffered saline PBS buffer systems.
TBS buffers should be used for the detection of phosphoproteins, in order to avoid interference from the phosphates in PBS buffers. TBS can also be used for the detection of alkaline phosphatase—conjugated secondary antibodies In some cases, a product supplier might specifically recommend PBS or TBS buffers for an antibody to obtain optimal results. At a minimum, results should be repeatable within and between Western blotting experiments 18 , 64 , and all samples should be run with replicates.
Replication of results in a different laboratory adds another level of confidence. It confirms the robustness of the antibody and the experiment itself, given the multitude of small differences that may exist between the methods, equipment, and reagents used Such unintentional variations such as minor differences in antibody dilution, antibody incubations, washes, and other aspects of the Western blotting procedure can be a substantial source of error.
Detailed reporting of experimental procedures is an effective way to reduce this type of error, with many peer-reviewed journals now encouraging this level of transparency.
Even so, it is still common for published studies to omit key details of the Western blotting procedure such as the type of detection performed enzymatic versus fluorescence and how the authors document results film exposure, timing of exposure, digital imaging, etc. Without such critical information, other researchers may be unable to reproduce the experimental results. Even so, it is still common for published studies to omit key details of the Western blotting procedure used.
Such information includes the system on which fluorescent detection was captured and the associated system settings. Similarly, for enzymatic detection it is important to report whether film or camera was used and what the exposure time was.
Tackling the current challenges and considerations in traditional Western blotting techniques is important, as is the need to look forward to other techniques and variations on this technique that will be of significant benefit to researchers in the future.
Some of these techniques may improve a number of parameters that are current challenges to the traditional technique and therefore could promote greater consistency of results, including multiplexing, advances in expression tags, and the detection of protein modifications. The future of Western blotting lies in harnessing the biological sample to the maximum capacity via multiplexed detection.
Although Western blotting is a staple assay of many labs, they still present several limitations. The assay can be manual, slow, and time-intensive during most steps and difficult to automate at scale. Electroblotting to a membrane and subsequent antibody incubations can also lead to substantial antigen loss Capillary electrophoresis and microfluidic Western blotting 66 represent two areas of innovation of the core assay concept.
Microfluidic Western blotting improves on the current process by both down-scaling size and increasing multiplexing, thus achieving detection of 10 separate proteins on one membrane with a fraction of the sample 67 , Capillary Western blotting through capillary gel electrophoresis CGE replaces the existing gel and membrane system with protein separation, immobilization, and staining in a single column CGE is considered to increase reproducibility of Western blottings, improve quantification, reduce hands-on time, and improve automation potential It is important to note that with the change in assay context, the behavior of antibodies also changes.
Therefore, direct comparison of protein expression and modification on a single membrane by multiplexing antibodies provides the best advancement in antibody validation for Western blotting. Spectrally-distinct fluorophores can be conjugated to primary antibodies or secondary detection antibodies to allow simultaneous detection on one blot. Here, domain-specific primary antibodies directed against the N and C termini of the target are used to analyze the overlap of staining and domain-specific bands.
This technique can also be used to analyze protein isoforms 20 , This method also enables analysis of protein modifications relative to the unmodified target.
Relative quantification of ubiquitination, sumoylation, glycosylation and acetylation are obtainable in this system 53 , — Current methods rely on detection of primary antibodies using spectrally-distinct fluorophores, either conjugated directly to the primary antibody or through binding of secondary antibodies.
Multiplexed immunoassays offer many advantages. These include more data points per sample, higher throughput, and in many cases reduced cost. Multiplexing also eliminates confounding variables such as … Read More ». Having claimed over four million … Read More ». Download PDF Protein tags underpin a broad range of research applications. These include affinity purification, where protein tags enable recombinant fusion proteins to be captured on a solid matrix, and … Read More ».
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