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Background

Antibodies are one of the commonly used reagents in life science research, diagnostics, and testing. Antibody validation is a key process to ensure the specificity, accuracy, and reliability of antibodies, which is of great significance for obtaining credible experimental results. With the development of life sciences, its importance has become increasingly prominent, as antibody quality directly affects the correctness of research conclusions and the rigor of the entire scientific research project. The International Working Group on Antibody Validation (IWGAV) published a paper, "A proposal for validation of antibodies," in the renowned journal Nature Method. The article argues that validating antibody specificity is currently the most important step to ensure antibody reproducibility, ensuring that the antibody can specifically recognize its intended target and maintain similar performance, so that all users operating according to the established experimental protocol can ultimately obtain the same results.

 

Conventional antibody validation methods include:

• Western Blot (WB)
• Immunocytochemistry (ICC)
• Immunohistochemistry (IHC)
• Immunoprecipitation (IP)
• Immunofluorescence (IF)
• Enzyme-linked immunosorbent assay (ELISA)
• Gene knockout or knockdown validation (e.g., CRISPR-Cas9, RNAi)
• Protein Chip technology
• Flow Cytometry (FC)
• Chromatin Immunoprecipitation (ChIP)

 

This topic will detail the experimental principles, procedures, and precautions of each method mentioned above.

Liman team provides a series of products and services for antibody validation: KO cell lysate for WB validation, IP validation ，KO cell lines for ICC validation, IF validation and FC validation etc.

 

I. Western Blot (WB)

Experimental Principle

Western blotting is a technique that transfers proteins from an electrophoresis gel to a solid support (such as a PVDF membrane or nitrocellulose membrane), and then uses specific antibodies to detect the target protein. First, the mixed protein sample is separated according to the size of the protein's molecular weight by SDS-PAGE electrophoresis. After electrophoresis, the protein is transferred from the gel to the membrane, so that the position of the protein on the membrane corresponds to that in the gel. Then, the non-specific binding sites on the membrane are blocked with a blocking solution to reduce background interference. Then, the primary antibody is added, which will specifically bind to the target protein. After washing away the unbound primary antibody, a secondary antibody labeled with an enzyme (such as horseradish peroxidase or alkaline phosphatase) or a fluorescent group is added, which will bind to the primary antibody. Finally, the target protein band is displayed through an enzyme substrate color reaction or fluorescence detection.

Experimental Steps

1. Protein sample preparation: Collect cells or tissues, add an appropriate amount of protein lysis buffer (containing protease inhibitors), lyse on ice, centrifuge to collect the supernatant, and determine the protein concentration.

2. SDS-PAGE electrophoresis: Prepare a polyacrylamide gel of appropriate concentration, mix the protein sample with the loading buffer, load the sample, perform electrophoresis, and set the appropriate voltage and time.

3. Transfer: Transfer the gel after electrophoresis and the PVDF membrane or nitrocellulose membrane in a transfer device. Wet transfer or semi-dry transfer methods can be used. Determine the transfer time and current according to the size of the protein molecule.

4. Blocking: Place the transferred membrane in a blocking solution (such as 5% skim milk or BSA solution) and incubate at room temperature for 1-2 hours.

5. Primary antibody incubation: Place the blocked membrane in a diluted primary antibody solution and incubate at 4℃ overnight or at room temperature for 2-3 hours.

6. Washing: Wash the membrane 3-5 times with TBST or PBST buffer, 5-10 minutes each time.

7. Secondary antibody incubation: Place the membrane in a diluted secondary antibody solution and incubate at room temperature for 1-2 hours.

8. Washing: Wash the membrane again 3-5 times with TBST or PBST buffer.

9. Color development or detection: If it is an enzyme-labeled secondary antibody, add the corresponding enzyme substrate for color development; if it is a fluorescent secondary antibody, detect it on a fluorescence imager.

Precautions

1. During protein sample preparation, ensure that the protein is fully lysed and avoid protein degradation. Fresh protease inhibitors can be added.

2. Pay attention to the choice of gel concentration during electrophoresis to ensure that the target protein is well separated.

3. During transfer, pay attention to the close contact between the membrane and the gel to avoid air bubbles, and optimize the transfer conditions according to the protein size.

4. The dilution ratio of the primary and secondary antibodies should be determined through preliminary experiments to avoid false positive or false negative results due to excessively high or low antibody concentrations.

 

II. Immunocytochemistry (ICC)

Experiment   Principle

Immunocytochemistry is a technique that uses antibodies to detect target antigens at the cellular level. After fixation and permeabilization, antibodies can enter the cells and bind to target proteins. Fixation maintains the morphological structure of cells, while permeabilization makes the cell membrane permeable to antibodies. Then, a primary antibody is added, which specifically binds to the target protein within the cells. A secondary antibody labeled with a fluorophore or enzyme is then added. Fluorescence signals are observed using a fluorescence microscope, or an enzyme substrate color reaction to determine the localization and expression of the target protein within the cells.

Experimental Steps

1. Cell Culture and Fixation: Cells are seeded on coverslips or culture dishes and cultured to the appropriate density. Cells are fixed with a fixative such as paraformaldehyde, generally for 15-30 minutes.

2. Permeabilization: Cells are treated with a permeabilizing agent such as 0.1%-0.5% Triton X-100 to allow antibodies to enter the cells. Treatment time is 5-15 minutes.

3. Blocking: Block non-specific binding sites on cells with blocking solution (e.g., 5% BSA or serum), incubate at room temperature for 30 minutes - 1 hour.

4. Primary Antibody Incubation: Add the diluted primary antibody to the cells, incubate overnight at 4°C or for 1-2 hours at room temperature.

5. Washing: Wash cells with PBS buffer 3-5 times, 5 minutes each time.

6. Secondary Antibody Incubation: Add fluorescently labeled or enzyme-labeled secondary antibody, incubate at room temperature for 30 minutes - 1 hour.

7. Washing: Wash cells with PBS buffer 3-5 times again.

8. Mounting and Observation: If using a fluorescently labeled secondary antibody, mount with an anti-fluorescence quenching mounting medium and observe under a fluorescence microscope; if using an enzyme-labeled secondary antibody, add the appropriate substrate to develop color and observe under an ordinary optical microscope.

Precautions

1. The choice of fixative and fixation time should be determined according to the cell type and target protein characteristics to avoid over-fixation that could damage antigenic epitopes.

2. The concentration and treatment time of the permeabilizing agent should be optimized to prevent excessive permeabilization affecting cell morphology and structure.

3. During primary and secondary antibody incubation, care should be taken to keep the cells moist to prevent drying.

 

III. Immunohistochemistry (IHC)

Experimental Principle

Immunohistochemistry is similar to immunocytochemistry, but the target antigen is in tissue sections. After tissue fixation, paraffin embedding, and sectioning, antigen retrieval is performed to expose masked antigenic epitopes. Then, primary and secondary antibodies are added sequentially, and enzyme substrate color reactions or fluorescence detection are used to observe the expression and distribution of the target antigen in the tissue, which can be used to study cell type identification and disease-related protein expression changes in tissues.

Experimental Steps

1. Tissue Fixation and Embedding: Fix tissue samples with fixatives such as paraformaldehyde, then perform dehydration, clearing, wax impregnation, and embedding steps to prepare paraffin blocks.

2. Sectioning: Cut paraffin blocks into thin sections (generally 3-5μm) and attach them to slides.

3. Antigen Retrieval: Select an appropriate antigen retrieval method, such as heat retrieval (boiling in citrate buffer or EDTA buffer) or enzyme retrieval (such as trypsin digestion), to expose antigenic epitopes, according to different antigens and tissue types.

4. Blocking: Block non-specific binding sites on sections with blocking solution (e.g., 5% BSA or serum), incubate at room temperature for 30 minutes - 1 hour.

5. Primary Antibody Incubation: Add diluted primary antibody to the sections, incubate overnight at 4°C or for 1-2 hours at room temperature.

6. Washing: Wash sections with PBS buffer 3-5 times, 5 minutes each time.

7. Secondary Antibody Incubation: Add enzyme-labeled or fluorescently labeled secondary antibody, incubate at room temperature for 30 minutes - 1 hour.

8. Washing: Wash sections with PBS buffer 3-5 times again.

9. Color Development or Detection: If using an enzyme-labeled secondary antibody, add the corresponding enzyme substrate for color development.

Precautions

1. Tissue fixation time should not be too long to avoid affecting antigenicity.

2. Antigen retrieval conditions should be strictly controlled, as different tissues and antigens may require different retrieval methods and times.

3. Section thickness should be uniform to avoid affecting staining results and observation.

 

IV. Immunoprecipitation (IP)

Experimental Principle

Immunoprecipitation (IP) is a biotechnology used to study protein-protein interactions. This technique treats proteins as antigens and utilizes the property of specific binding between antibodies and antigens. The target protein is isolated by binding it to a specific antibody, which then binds to Protein A/G agarose beads, forming a complex that is separated via centrifugation. The main principle is: antigen-antibody binding to form an immune complex, Protein A/G binds to the antibody Fc region, separating the complex containing the target protein via centrifugation.

Experimental Steps

1. Sample Preparation: Collect cell or tissue samples, add an appropriate volume of lysis buffer (RIPA buffer), incubate on ice for 30 minutes, centrifuge at 12,000g for 15 minutes, and collect the supernatant. 2. Pre-treatment: Take an appropriate amount of Protein A/G agarose beads, wash 2-3 times with lysis buffer, add to the sample supernatant for pre-treatment (remove non-specific binding). 3. Immunoprecipitation: Add specific antibody to the pre-treated supernatant, incubate overnight at 4°C, add pre-treated Protein A/G agarose beads, incubate at 4°C for 2-4 hours. 4. Washing and Elution: Centrifuge at 3000g to collect the precipitate, wash 3-4 times with lysis buffer, add an appropriate amount of loading buffer, boil at 95°C for 5 minutes, centrifuge to collect the supernatant. 5. Detection and Analysis: Perform SDS-PAGE electrophoresis and Western Blot to detect the target protein.

Precautions

1. Sample Preparation: All operations should be performed at 4°C or on ice as much as possible. Add protease inhibitors to prevent protein degradation and avoid repeated freeze-thaw cycles. 2. Antibody Selection: Use highly specific antibodies. Confirm the antibody's binding ability with Protein A/G and optimize antibody concentration. 3. Experimental Conditions: Strictly control temperature conditions, optimize incubation time, and select appropriate washing conditions. 4. Key Controls: Set up negative controls (without antibody or using isotype control antibody) and Input samples (total protein without IP treatment) to avoid interference from IgG heavy and light chains.

 

V. Immunofluorescence (IF)

Experimental Principle

Immunofluorescence uses fluorescently labeled antibodies to detect target antigens. The location and expression of the antigen are determined by observing the fluorescent signal under a fluorescence microscope. Similar to immunocytochemistry and immunohistochemistry, after cells or tissues are fixed and permeabilized, a specific primary antibody is added, followed by a fluorescently labeled secondary antibody. The fluorophore emits fluorescence under excitation by light of a specific wavelength, allowing for direct observation of the target protein's distribution in cells or tissues.

Experimental Steps

1. Cell or Tissue Processing: Same as the pretreatment steps for immunocytochemistry or immunohistochemistry, including fixation, permeabilization (cells), and sectioning (tissues).

2. Blocking: Block non-specific binding sites with blocking solution.

3. Primary Antibody Incubation: Add the diluted primary antibody; incubation conditions are as described above.

4. Washing: Wash thoroughly with PBS buffer.

5. Secondary Antibody Incubation: Add the fluorescently labeled secondary antibody. Note: Perform this step in the dark and incubate at room temperature for an appropriate time.

6. Washing: Wash again to remove unbound secondary antibody.

7. Mounting and Observation: Mount with an anti-fluorescence quenching mounting medium and observe under a fluorescence microscope. Select the appropriate excitation wavelength and filter.

Precautions

1. Avoid light exposure throughout the experiment, especially during secondary antibody incubation and storage, to prevent fluorescence quenching.

2. Different fluorophores have different excitation wavelengths; select the appropriate microscope filter for observation.

 

VI. Enzyme-Linked Immunosorbent Assay (ELISA)

Experimental Principle

ELISA is a detection technique based on the specific binding of antigen-antibody and the colorimetric reaction catalyzed by enzymes. The antigen or antibody is immobilized on a solid-phase carrier (such as a microplate). The sample is added, and the target antigen or antibody in the sample binds to the immobilized antigen or antibody. Then, an enzyme-labeled secondary antibody or antigen is added. After incubation and washing, an enzyme substrate is added, and the enzyme catalyzes a colorimetric reaction. The target antigen or antibody concentration is quantitatively detected by measuring the absorbance value.

Experimental Steps

1. Coating: Dilute the antigen or antibody with coating buffer and add it to the microplate. Incubate overnight at 4°C or for 2-4 hours at room temperature, then wash 3-5 times with washing solution.

2. Blocking: Add blocking solution, incubate at room temperature for 1-2 hours, and wash again.

3. Sample Addition: Add the sample or standard, incubate at room temperature for 1-2 hours, and wash.

4. Primary or Secondary Antibody Incubation: For indirect methods, add the primary antibody and incubate, then wash; for sandwich methods, add the enzyme-labeled secondary antibody and incubate, then wash.

5. Substrate Color Development: Add the enzyme substrate solution, incubate at room temperature in the dark for a certain time, and add the stop solution after the color change is obvious. 6. Absorbance Measurement: Measure the absorbance of each well using a microplate reader and calculate the concentration of the target antigen or antibody in the sample based on the standard curve.

Precautions

1. Select an appropriate microplate with a uniform and smooth surface to ensure effective coating of the antigen or antibody.

2. The washing process must be thorough to avoid residual substances affecting subsequent reactions.

3. Store and use the enzyme substrate according to the instructions to prevent substrate failure.

 

VII. Gene Knockout or Knockdown Verification (e.g., CRISPR-Cas9, RNAi)

Experimental Principle

The CRISPR/Cas9 RNP gene editing method involves mixing multiple in vitro chemically synthesized single-guide RNAs (sgRNAs) corresponding to the target gene with Cas9 protein in vitro to form a ribonucleoprotein complex (RNP). The RNP is delivered into cells via electroporation/lipid transfection, similar to a "transient" transfection process. This avoids the resistance screening process required for traditional lentivirus/plasmid methods, resulting in highly efficient and stable genetically modified cell pools for gene knockout. RNAi uses double-stranded RNA (dsRNA) or small interfering RNA (siRNA) to bind to and induce the degradation of target mRNA, thereby reducing the expression level of the target gene. In antibody validation, after knocking out or knocking down the target gene, the change in the expression of the corresponding protein is detected. If the antibody cannot detect or the expression is significantly reduced, it indicates that the antibody is highly specific; otherwise, non-specific binding may exist.

Experimental Steps (Example: CRISPR/Cas9 RNP Gene Editing Method):

1. sgRNA Design and Synthesis: Design and synthesize specific sgRNA sequences based on the target gene sequence.

2. Purchase or prepare and purify Cas9 protein.

3. Cell Transfection: Transfect sgRNA and Cas9 protein into target cells using methods such as lipid transfection or electroporation.

4. Knockout Efficiency Identification: After transfection, continue cell culture and identify gene knockout using methods such as PCR and sequencing.

5. Protein Detection: Extract cell protein and perform Western Blot or other detection using the target antibody to observe changes in protein expression.

Precautions

1. sgRNA design should ensure specificity and avoid off-target effects.

2. Optimize cell transfection efficiency to improve the success rate of gene knockout.

3. The knockout efficiency verification process must be rigorous to ensure accurate gene knockout results.

 

VIII. Protein Chip Technology

Experimental Principle

Protein chips are microarrays formed by immobilizing a large number of different proteins or antibodies on a solid-phase carrier surface. After incubation with the sample to be tested, the target protein in the sample will bind to the corresponding antibody or ligand on the chip. Then, the binding signal is detected through labeling techniques (such as fluorescence labeling, enzyme labeling, etc.), thus simultaneously detecting the expression levels, interactions, and other information of multiple proteins.

Experimental Steps

1. Chip preparation: Spot different proteins or antibodies onto the surface of a solid-phase chip for immobilization and blocking.

2. Sample preparation: Label (e.g., fluorescence labeling) or biotinylate the sample to be tested so that it can be detected.

3. Chip hybridization: Incubate the treated sample with the chip under appropriate conditions to allow the target protein to bind to the corresponding molecule on the chip.

4. Washing: Wash away unbound substances with washing solution.

5. Detection: According to the labeling type, use a fluorescence scanner or enzyme-linked immunosorbent assay (ELISA) reader to detect the signal on the chip and analyze the data.

Precautions

1. The amount of protein or antibody spotting and the immobilization conditions during chip preparation must be precisely controlled to ensure the quality and reproducibility of the chip.

2. Appropriate sample processing should be used to avoid the labeling process affecting protein activity and structure.

 

IX. Flow Cytometry Flow Cytometry ）

Experimental Principle

Flow cytometry is a technique for rapid quantitative analysis and sorting of suspended cells. Cells are made into a single-cell suspension. After staining with fluorescently labeled antibodies, in a flow cytometer, cells pass through a laser beam one by one. The laser excites the fluorophores to emit fluorescence. The flow cytometer detects the cell's scattered light signal (reflecting cell size and internal structural complexity) and fluorescence signal (reflecting the expression of the target protein), allowing for analysis and sorting of cell populations.

Experimental Steps

1. Cell preparation: Collect cells, wash with PBS buffer to prepare a single-cell suspension, count, and adjust cell concentration.

2. Antibody staining: Incubate cells with fluorescently labeled antibodies on ice in the dark for a certain period of time, then wash with PBS buffer.

3. Fixation (optional): Fix cells with a fixative if necessary.

4. Instrument detection: Add the stained cell suspension to the flow cytometer, set appropriate detection parameters, collect data, and perform analysis.

Precautions

1. The cell suspension must be prepared in a single-cell state to avoid cell clumps affecting the detection results.

2. When performing antibody staining, pay attention to the amount of antibody and incubation time, and perform the operation in the dark.

3. The parameters of the flow cytometer should be optimized according to the cell type and detection target.

 

X. Chromatin Immunoprecipitation (ChIP)

Experimental Principle

Chromatin immunoprecipitation is used to study the interaction between proteins and DNA in vivo. First, cells are treated with crosslinking agents such as formaldehyde to crosslink and fix proteins and DNA. Then, the chromatin is sonicated into small fragments, and DNA fragments bound to the target protein are immunoprecipitated using specific antibodies. After decrosslinking and purification, the precipitated DNA is subjected to PCR amplification or sequencing analysis to determine the DNA sequences bound to the target protein.

Experimental steps:

1. Crosslinking: Add formaldehyde during cell culture to crosslink proteins and DNA, then terminate the crosslinking reaction with glycine.

2. Cell lysis and chromatin preparation: Collect cells, lyse cells, isolate chromatin, and sonicate chromatin into appropriately sized fragments using a sonicator.

3. Immunoprecipitation: Add specific antibodies, incubate at 4°C overnight, then add Protein A/G magnetic beads or agarose beads, and continue incubation to allow the antibody-antigen-DNA complex to bind to the beads.

4. Washing: Wash the beads sequentially with low-salt, high-salt, and LiCl buffers to remove non-specifically bound substances.

5. Decrosslinking: Resuspend the beads in TE buffer, add NaCl for decrosslinking, and incubate at 65°C overnight.

6. DNA purification: Purify DNA using methods such as phenol-chloroform extraction and ethanol precipitation.

7. Detection: Perform PCR amplification or sequencing analysis on the purified DNA.

Precautions:

1. The crosslinking time and formaldehyde concentration should be controlled to avoid over-crosslinking or under-crosslinking.

2. Optimize the sonication conditions to ensure that the chromatin fragments are of the appropriate size.

3. The specificity and amount of antibodies in the immunoprecipitation process should be appropriate, and the washing of the beads should be thorough.

 

Through an in-depth understanding of these antibody verification methods, researchers can choose appropriate verification methods according to their experimental needs and research objects, thus laying a solid foundation for high-quality research results. In practical applications, the combined use of multiple verification methods can often more comprehensively and accurately evaluate the performance of antibodies, allowing us to move forward more steadily on the path of exploring the mysteries of biology.

 

The Liman team provides a series of products and services for antibody verification: KO cell lysates are used for WB verification and IP verification, and KO cell lines are used for ICC verification, IF verification, and FC verification, etc.