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What aspect of primary antibodies would you like to inquire about?
You’ll have to first specify the antigen that you’re looking for and what tissue or cell it is located in, and then consider the antibody’s host species. Choose a suitable antibody based on the experimental technique. The various experimental techniques can require antibodies of various properties. The datasheet of each antibody typically gives its recommended experimental uses, so make sure that your antibody is confirmed and suitable for your method. If the antibody is being applied, read the product datasheet to determine whether the selected primary antibody is approved for your experimental environment. In the absence of confirmation, do preliminary experiments to validate its efficacy.
The average of all primary antibodies will be the following dilutions: 1:100 - 1:1000 as reported on their datasheet. But in practice you can dilution according to your experiment and get the working concentration that is the best.
If the staining is too weak, you can increase the concentration of the antibody. Conversely, if there is excessive background staining, you should decrease the concentration.
If possible, repeat several dilution experiments in the recommended ratio to determine the ideal dilution for your experimental setup. For example, if a datasheet recommends 1:2000 dilution, you could dilution it 1:1200, 1:500, 1:1000, 1:2000, etc. to see which is best with least noise.
Stock unused primary antibodies in a refrigerator between 2-8°C. You can aliquote them and store at -20°C or -80°C to use them for long term storage. Do not freeze-thaw on and off to keep them going. You can also aliquot them into single-use amounts based on experimental needs to reduce the impact of multiple openings on their activity. To aliquot, be sure each aliquot is 10 μL or less. Because of liquid evaporation and container adsorption, the smaller the volume of each aliquot, the higher the effect on antibody concentration. Therefore, it is advisable to balance the aliquot size with the need for minimal exposure to air and repeated handling.
First, we confirm that our product is sealed properly. However, during transportation and storage, a small amount of antibody may remain adhered to the walls or lid of the container. You can remove the solution that has attached to the walls by briefly centrifuging the sample.
(a) Optimize Antibody Concentration:
If you use a high amount of primary or secondary antibodies, then you risk nonspecific binding. Find out the right dilution ratio with initial experiments for a good working concentration. Reduce the concentration of the first antibody during the experiment until the signal-to-background ratio is best.
Also, don’t overincubate the antibody; longer incubation causes more background. You can put a timer in so you remember to finish the incubation on time.
(b) Blocking Step:
Before incubating with the primary antibody, block the sample using a blocking agent (such as normal serum, BSA, or nonfat dry milk). This effectively prevents the primary antibody from binding to nontarget structures, thereby reducing false-positive signals.
Choose a blocking agent with low nonspecific binding that is not from the same species as the primary antibody. For instance, when you are employing a rabbit primary antibody, select mice or other species serum as the blocking agent so that the blocking solution will not be cross-reactive with the antibodies in it.
(c) Use Appropriate Wash Buffers:
Between each step wash thoroughly in wash buffers of a proper quality (PBS or TBS) to remove loose primary antibodies and background signals. Nonspecific binding can also be lowered by mixing wash and stain buffers with a nonionic detergent (Triton X-100, Tween 20).
(d) Select Appropriate Primary Antibodies:
Monoclonal antibodies typically exhibit higher specificity, which helps reduce nonspecific binding. Whenever possible, choose validated primary antibodies with strong literature support. When selecting a primary antibody, review data provided by suppliers, including user reviews and relevant literature citations, to ensure reliable performance.
Make sure your primary antibody and secondary antibody are from distinct species so you don’t cross react. So if you’re working with a rabbit primary antibody, for example, you’d use a mouse secondary antibody. This way the detection is more precise and sensitive.
There are several reasons why no bands may appear in your Western blot results. You can refer to the following factors to identify the issue:
(a) Sample Issues:
Target Protein Absence: Ensure that the sample indeed contains the target protein. You can confirm its presence using other methods, such as ELISA or immunohistochemistry.
Improper Sample Handling: Protein degradation or loss may occur during sample extraction. Use fresh samples, maintain low temperatures during handling, and add protease inhibitors to prevent degradation.
(b) Transfer Efficiency:
Incomplete Transfer: Incorrect settings for current, time, or temperature during transfer may prevent effective transfer of the target protein to the membrane. It is recommended to use reversible staining to verify transfer efficiency.
Incorrect Membrane Choice: Select a membrane with the right pore size, based on the molecular weight of the target protein.
(c) Antibody Issues:
Unsufficient Strength of Primary or Secondary Antibodies: Unsustained weak or no signal is possible when antibody strength is too low. Be sure to check the antibody dilution ratios first by performing preliminary experiments, in order to get them within the recommended range.
Failure to Use Specific Antibody: The antibodies employed might be unspecific for the target protein and may cause undetectable signals. Select validated primary antibodies and check relevant literature supporting their performance.
(d) Washing and Blocking Steps:
Inadequate Washing: Insufficient washing steps can lead to high background signals that obscure the target band. Ensure thorough washing between each step to remove unbound antibodies.
Insufficient Blocking: Incomplete blocking can lead to nonspecific binding, increasing background signals. Use appropriate blocking agents (such as BSA or nonfat dry milk) and ensure thorough blocking.
(e) Experimental Conditions:
Improper Exposure Time: If the exposure time during imaging is too short, signals may not be captured. Adjust the exposure time appropriately for optimal image quality.
Poor Electrophoresis Conditions: Overloading samples or running electrophoresis for too long can affect band clarity and visibility. Follow recommended loading conditions and control electrophoresis time.
(f) Detection Reagent Issues:
Expired Detection Reagents: Ensure that fresh detection reagents are used and that they have not expired or become contaminated, especially ECL detection reagents. If detection reagents are inactive, visible signals will not be produced.
There are several reasons why IHC results may not be clear, primarily including the following aspects:
(a) Antibody-Related Factors:
Underconcentration of the Antibody: If the concentration of the antibody is underconcentrated, then there will be faint staining signals. It’s suggested to figure out the best antibody dilution through pilot experiments.
Quality Concerns with Antibodies: Expired or degraded antibodies affect staining. Make sure antibodies are past their expiry date and properly stored.
Lack of Accurate Antigen Retrieval: Some antigens need certain retrieval operations to reveal their epitopes. Too little retrieval and antibodies don’t successfully interact with the target antigen.
(b) Experimental Operation Issues:
Insufficient Washing: if washing after antibody incubation does not completely flush out unbound antibodies, then background staining is possible and will change the signal clarity and brightness. This can be alleviated by increasing the number of washes or washing longer.
Too Short DAB Staining Time: Insufficient DAB staining reaction time can also result in unclear staining. It is generally recommended to terminate the reaction immediately upon observing brown coloration to avoid overstaining.
Improper Handling of Sections: If sections are not handled properly during deparaffinization and rehydration (e.g., excessive soaking time), it may impact staining results. Ensure that sections are well-dried and processed under appropriate conditions.
(c) Tissue Sample Issues:
Low Expression Levels of Target Antigen: If the expression level of the target antigen in the tissue sample is inherently low, it may lead to faint staining results. Comparing with a positive control sample known to contain the target antigen can help confirm this issue.
Improper Tissue Fixation: Excessive temperature or prolonged fixation time during the fixation process can damage antigen epitopes, resulting in weakened staining signals. Therefore, selecting appropriate fixatives and fixation conditions is crucial.
The causes of high background in immunoprecipitation experiments can be several:
Lack of Concentration of Antibody: Too much antibody makes a nonspecific binding and adding noise to it. It is advisable to optimize the antibody concentration to reduce background.
False Lysis Buffer Formulation: If the lysis buffer contains too many detergents or has a harsh formula, nonspecific proteins may be released. Lower the detergent strength or use a weaker detergent.
Unreliable Washing: Inadequate washing after immunoprecipitation could leave loose nonspecific proteins floating around in the final sample leading to more background signals.
Insufficient Blocking: If the membrane is not adequately blocked before immunoprecipitation, it may lead to increased background signals. Consider performing a blocking step before incubating with the primary antibody.
Poor Antibody Specificity: The antibodies used may also bind to proteins other than the target protein, resulting in elevated background signals. Selecting more specific antibodies or optimizing the antibodies can help reduce this issue.
An isotype control is an immunoglobulin that is of the same species origin, subtype, and dosage as the primary antibody used in the experiment, but it does not bind to the target antigen. Its primary purpose is to help distinguish between specific signals and nonspecific background signals.
Typical tags are fluorescent (visualisable), enzymes (e.g., horseradish peroxidase HRP, substrate reactions) and biotinylation (signal amplification). A label should be based on the experimental use and the level of signal.
These antibodies labels are enzyme labeling, biotin streptavidin labeling, fluorescent labelling, nanoparticle conjugation and oligonucleotide conjugation that we are currently able to offer. For more details, please refer to our custom antibody labeling service.
We also offer a full complement of tagged antibodies such as peptide and polypeptide tag antibodies, fluorescent tag antibodies, and hapten antibodies. We discuss all of our products related to them in detail at the Tag Antibodies page.
In addition to full antibodies, we also offer antibody fragments, including Fab and F(ab')2 fragments. You can explore our Custom Antibody Fragmentation for more information.
When selecting a tag, the following factors should be considered:
Type of Experiment: Various Experiments (flow cytometry, immunohistochemistry, etc.) have varying requirements for tags. Fluorescent dyes are good for experiments where the image is wanted, but enzyme tags are good for experiments where the chemical reaction is wanted.
Signal Strength and Stability: Pick tags that are signal strong and stable so the target is clearly visible in the experiment.
Cross-Reactivity: Make sure the tag you chose does not cross-react with other parts of the experiment as it might change the results.
Creative Diagnostics is a "One stop" antibody solutions provider serving the pharmaceutical, biotech, diagnostic and university research organizations around the world.
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