IHC, WB, ELISA

How to Design Positive and Negative Controls

Using appropriate scientific controls in experiments is essential for validating research findings. Scientific controls serve to minimize the effects of variables other than the independent variable. By setting reference points for the treatment group to compare data against, the scientific control group helps eliminate alternate explanations of experimental results, especially uncontrolled bias and systematic error, and helps validate the performance of the experimental set-up and increases the reliability of the results.

Positive and Negative Controls

Two main types of controls must be included in an experimental plan - positive and negative. Positive controls are used to assess the test validity of the experimental protocol or equipment by producing the expected result. Negative controls are characterized by the absence of reagents or components that are necessary for successful analyte detection. By not exposing the negative control group to the experimental treatment or any other treatment, they are not expected to produce any result due to any variable in the experiment and may serve as the “baseline”.

Table 1. Controls & Treatment Group Outcome Interpretation

Positive Control Negative Control Treatment Group Outcome Interpretation
+ + -
  • False-positive
  • Possible causes: use of inappropriately high antibody concentration, non-specific antibody-antigen binding, buffer components, etc.
- + -
  • False-negative (buffer components, etc.)
  • Protocol requires optimization
+ - -
  • Treatment had no effect
  • The procedure is working and optimized
  • Negative results are valid (true negative)
+ - +
  • Treatment had produced an effect
  • The procedure is working and optimized
  • Positive results are valid (true positive)
+ + +
  • A positive result may be due to false-positive or non-specific signal
  • A confounding variable is involved in the phenomenon under study
  • Positive results are not solely due to the treatment

Apart from positive and negative controls, there are also many types of controls specific to the type of experiment being performed. The selection and use of proper controls will ensure experimental results are valid and save valuable money and time. As a result, including controls in experiments is a standard practice in research and diagnostic investigations. Below, we summarize essential positive and negative controls for immunohistochemistry, western blot, and ELISA protocols.

Immunohistochemistry Controls

Running appropriate controls in IHC staining experiments is critical to confirm the validity of the observed staining pattern, facilitate consistent performance, and ensure accurate interpretation of the results. Several types of established controls can be used to support the specificity of IHC results as shown in the table below.

Table 2. Established Controls for IHC

Types of IHC Controls Content Purpose
Antigen (tissue) controls Positive control
  • Qualitative: tissue section known to express the target protein
  • Quantitative: tissue section known to express the target protein within the desired range
  • Demonstrate that the staining protocol is successfully performed and giving the expected level of sensitivity/specificity as characterized during technical optimization
  • Confirms that negative results are accurate
Negative control Tissue section known not to express the target protein Check for non-specific signal and false-positive results
Autofluorescence of endogenous tissue background staining control Tissue section before applying the primary antibody
  • Certain tissues such as those rich in elastin, collagen and lipofuscin have inherent properties (endogenous fluorescent molecules) that could result in the background and affect result interpretation (false positive)
  • Observe samples under the microscope using either fluorescence (for fluorescent labels) or bright-field (for chromogenic labels) illumination before every staining experiment to ensure there is no signal inherent to the tissue itself
Reagent controls No primary antibody control (secondary antibody only control) Tissue section incubated with the antibody diluent alone and no primary antibody, followed by incubation with secondary antibodies and detection reagents (same staining protocol) Ensures staining is produced from detection of the antigen by the primary antibody and not by the detection system or the specimen (non-specific binding)
Isotype control Tissue section incubated with the antibody diluent and a non-immune antibody of the same isotype, at the same concentration as the primary antibody, followed by incubation with secondary antibodies and detection reagents
  • Ensures that the observed staining is not caused by non-specific interactions of the antibody with the tissue
  • Any background staining observed with this control should be negligible and distinct from specific staining
  • Only used for controlling monoclonal primary antibodies
  • Matching isotype control properties with the primary antibody:
    • Same host species, isotype, and subclass
    • Same conjugation format
    • Equal conjugate-to-antibody ratios
Absorption control Tissue section incubated with pre-absorbed antibody instead of the primary antibody, followed by incubation with secondary antibodies and detection reagents (same staining protocol)
  • Inhibition of staining via adsorption of the primary antibody with the purified antigen/immunogen before usage
  • Demonstrate that the primary antibody binds specifically to the target antigen
  • Little or no staining is expected
  • More reliable if the immunogen is a peptide, as antibody-protein immunogen mixtures may themselves cause high background staining in tissues due to non-specific protein-tissue interactions

Western Blot Controls

Often considered the gold standard for protein detection and quantification, there are several types of established controls essential to support the specificity and validity of Western Blot analysis—as shown in the table below.

Table 3. Established controls for Western Blot

Types of WB Controls Characteristics
Positive control lysate Lysate from a cell line or tissue sample (from a tested species) known to express the target protein
  • Demonstrate that the staining protocol is successfully performed and giving the expected level of sensitivity/specificity as characterized during technical optimization
  • Confirms that negative results are accurate
  • Commonly used positive controls:
    • Samples from cells exhibiting overexpression of target protein
    • Cell line/tissue/experimental condition with a proven positive signal
    • Purified recombinant protein
Negative control lysate Lysate from a cell line or tissue sample known not to express the target protein
  • Check for non-specific binding (false-positive results)
  • Commonly used negative controls:
    • Samples from knockdown or knockout tissue/cell lines
    • Samples from RNA interference targeted lines
    • Cell line/tissue/experimental condition with proven negative signal
Loading control Antibodies to housekeeping proteins, or proteins that are expressed at equivalent levels in almost all tissues and cells
  • Semi-quantification of protein levels between wells
  • Confirm equal amount protein sample loaded and gel-membrane transfer efficiencies on all lanes during the procedure to validate that the observed differences in the target protein levels are not due to errors while loading/incubating the gel
  • Recommendation for suitable loading control:
    • Substantially different molecular weight to be easily distinguished from the target protein, avoid overlapping staining and interference of results
    • Highly expressed in the sample for dynamic ranges with the target protein
    • Housekeeping genes to ensure consistent expression under test conditions across all lanes
Endogenous control lysate A positive endogenous control lysate known to express the target protein
  • Essential when testing a sample of recombinant protein due to several possible difficulties with the expression of recombinant proteins, such as misfolding of recombinant protein from the endogenous native form that prevents antibody-epitope binding. This is particularly the case with tagged proteins.
  • Demonstrate that the staining protocol is successfully performed and giving the expected level of sensitivity/specificity as characterized during technical optimization and indicate whether there might be an issue with the recombinant protein
No primary antibody control The primary antibody is not added to one strip of the membrane, (only using antibody dilution buffer containing no antibody)
  • The secondary antibody is incubated on the sample in the same way as usual
  • Check if any non-specific binding (false positives) may be due to non-specific binding of the secondary antibody

Table 4. Commonly used loading controls for WB

Sample Type Protein Molecular Weight
Whole-cell / cytoplasmic protein β/α-actin 43
β/α-tubulin 55
GAPDH 30-40
Cyclophilin B 21
Vinculin 116
Mitochondria VDCA1/porin 31
Cytochrome c oxidase 16
HSP60 60
COX1 17
Nuclear proteins Lamin B1 66
TATA-binding protein TBP 38
PCNA 29
Histone H3 18
HDAC 60
Serum Transferrin 77
Muscle SDHA 73
Yeast Phosphoglycerate kinase 45
Plant tissue LHCP 25
APX3 32

ELISA Controls

To verify that the assay is performing accurately, below are various types of control samples you can use when running an ELISA.

Table 5. Established controls for ELISA

Types of ELISA Controls Characteristics
Positive Control Use either an endogenous soluble sample known to contain the target protein or a purified protein or peptide known to contain the immunogen sequence for the antibody used
  • Demonstrate that the staining protocol is successfully performed and giving the expected level of sensitivity/specificity as characterized during technical optimization
  • Confirms that negative results are accurate
Negative Control Lysate from a cell line or tissue sample is known not to express the target protein
  • Check for non-specific signal and false-positive results
  • Commonly used negative controls for ELISA: Zero blank & chromogen blank
  • OD>0.25 (ELISA dependent) for zero blank means high background
  • Comparing zero blank and chromogen blank helps to find the likely cause
  • Chromogen blank is useful when the background reading is caused by reservoir, metal, or chromogen
Standard Control Contains a known concentration of the target protein
  • Can be classified as a positive control, necessary for quantification of the experimental results and may also be useful for optimization
  • Gives the information necessary to make a standard curve. The R2 value of the trend line should be >0.99 as an unsatisfactory standard curve can be indicative of poor antibody binding or inadequate specificity for the target protein
Spike Control A known amount of standard diluted in serum from the species tested
  • Compared with the standard diluted in the normal diluent buffer to ensure there is nothing present in the matrix that interferes with the assay
  • Indicate assay performance, demonstrate that target protein is recoverable after being spiked into a matrix by calculating recovery percentage from the ELISA readout
  • Acceptable results are 80–120%
Endogenous Positive Control Endogenous wild type versions of the target protein
  • Essential when testing a recombinant protein sample
  • Always ensure the recombinant protein includes the immunogen sequence of the antibody you are using
  • Important to validate the results, as well as to indicate how well the reagents (eg antibodies) and procedure are working

Select & Validate Antibodies for your Application

Choosing the most appropriate antibodies for your research is critical for identifying the source of problems and validating results since including appropriate controls can save you time and frustration down the road. However, with varying validity information and the quality of commercially available antibodies, finding an antibody that works well for a specific application can be a challenge. We have compiled a 5-step guideline to help simplify the process of identifying high-quality antibodies for your research.

Table 6. Guidelines for selecting & validating antibodies

Step
1 Use search engines to find and compare available antibodies
  • All vendors supply “product information sheets” describing the antibody, its target protein target, and perhaps its clinical applications
  • Check the antibody datasheet (Swiss-Prot, Omnigene database, GeneCards entry) for the protein, which will usually provide relative levels of expression in various tissues
  • Boster provides gene infographics that display the basic information you need to know for each gene, covering almost all genes in human and mouse. Share them with your colleagues and friends!
  • Several search engines allow you to find and/or compare antibodies from different vendors
  • Recommended antibody resource websites:
2 Match the antibody type to your application
  • Each type of antibody has its advantages and disadvantages
  • Polyclonal antibodies are suitable for proteins with post-translational modifications or heterogeneity in structure or sequence, proteins present at low concentrations, or applications that require fast binding to a protein of interest. However, they show relatively high batch-to-batch variability
  • Monoclonal antibodies are highly specific and reproducible, making them an ideal choice for IHC applications. They ensure greater batch-batch homogeneity but are more challenging to work with when looking at low-abundance proteins or proteins that show variability, such as those with posttranslational modifications, in the epitope recognized by the antibody
  • Recombinant antibodies are more expensive but guarantee high batch-to-batch consistency
  • Doing quick literature research on PubMed to see which tissues and cells express the protein of interest will also be very useful
3 Buy from companies that will work with you
  • Choose a vendor who is willing to help you troubleshoot if an antibody does not perform as expected
  • Boster provides comprehensive troubleshooting advice that can save you a lot of precious time and help you avoid common mistakes. Download our free troubleshooting handbooks for IHC, Western blot and ELISA.
  • Look for antibodies with complete validation data recommended for your species and application
  • If you need assistance, please contact us at [email protected] and we will help you resolve your specific trouble.
4 Review publications, examine antibody data and references There are a number of published studies detailing their results and references for further validation of antibodies for your specific applications.
5 Validating antibodies for your application
  • Antibodies may perform differently due to various errors (packaging error, production errors, different antibody lot, etc.)
  • Optimize protocols for your specific applications.
  • Assess the specificity, sensitivity, and reproducibility of each antibody using the same buffers, sample types, and experimental conditions