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03 December, 2023 by Anshul (neobio)
Do your research findings often leave you puzzled due to the unpredictability of the outcomes? The answer may well lie in your use of monoclonal antibodies. Welcome to our comprehensive ‘Monoclonal Antibodies Troubleshooting Guide,’ a thoughtfully put together resource designed for researchers like you, encountering obstacles in their work with these molecular tools.
Monoclonal antibodies are the workhorses in research labs, used in a broad range of applications – from Immunohistochemistry and Flow Cytometry to Western Blotting and Immunofluorescence. At NeoBiotechnologies, we understand the critical role these antibodies play in precision research; however, their efficiency can be compromised by various issues such as non-specific staining, high concentration, and background staining in immunohistochemistry. Imperfections in implementing the monoclonal antibody can cause data inconsistency, rendering the results unfit for meaningful interpretation. Knowing how to troubleshoot these irregularities is key to obtaining accurate and reliable results.
For busy researchers like you, troubleshooting monoclonal antibody issues doesn’t need to be a complex task. It is a beneficial and practical process that can substantially improve the accuracy and reliability of your research outcomes.
Each monoclonal antibody is unique, and what works for one may not necessarily apply to another. Bear in mind these three common trouble spots:
Addressing these three areas often eliminates most of the standard issues arising in your monoclonal antibody utilization.
In the forthcoming sections of our guide, we will uncover the nuances, common problems, and troubleshooting techniques for optimizing the use of monoclonal antibodies. So, join us, as we delve deeper into solutions that will help ensure your research with monoclonal antibodies is accurate, reliable, and hassle-free.
In research, even the most experienced professionals may encounter issues with monoclonal antibody usage. At NeoBiotechnologies, we understand these challenges and offer solutions based on our four decades of experience in the biotech industry.
Non-specific staining can often occur in Immunohistochemistry (IHC) and lead to unusable data. This problem is primarily caused by two factors: improper sample preparation and antibody problems.
Inadequate or improper sample preparation can lead to the antibodies binding to proteins other than the target protein. It’s crucial to follow strict protocols for tissue fixation and preparation to ensure specificity.
The choice of antibodies, whether primary or secondary, can significantly influence the quality of staining. It’s important to use highly validated, monospecific antibodies, such as the Rabbit Recombinant Monoclonal Antibodies we produce at NeoBiotechnologies.
Using a high concentration of primary antibodies can increase nonspecific binding and background staining, leading to poor results. The solution lies in optimizing the antibody concentration. Start with the recommended dilution and adjust as necessary based on your results.
Background staining in IHC can be due to nonspecific antibody binding or endogenous peroxidase activity.
This is more common with polyclonal antibodies. The use of highly specific monoclonal antibodies, like the ones we manufacture, can minimize this issue.
This is particularly problematic in tissues with abundant hematopoietic elements. Using an appropriate peroxidase block can prevent this issue.
There are several strategies to reduce non-specific staining in IHC. These include:
Over-fixation can cause high levels of background staining. It’s important to optimize the fixation time for your specific sample type.
Decreasing the thickness of tissue sections can help reduce background staining.
Ensure complete removal of paraffin from tissue sections before staining.
Using a suitable blocking agent can prevent nonspecific binding of antibodies.
Select antibodies that are highly validated and specific to your target protein.
Use peroxidase and biotin blocking steps to reduce background staining due to endogenous peroxidases and biotin.
At NeoBiotechnologies, we are committed to providing reliable solutions for your research needs. Our team of experts, led by Dr. Atul K. Tandon, is here to assist you in your journey towards successful monoclonal antibody usage.
In addition to the basic troubleshooting methods, there are some advanced techniques that can help you ensure the maximum effectiveness of your work with monoclonal antibodies.
A lack of antigen could be a possible reason for the failure of antibody staining. In such situations, the protein expression can be checked by in situ hybridization. This technique uses a labeled complementary DNA or RNA strand to localize the presence of a specific DNA or RNA sequence in a tissue section. In some rare cases, translation may be blocked even though mRNA is detected. Hence, it’s essential to validate the presence of the protein to ensure that the antibodies can bind to their intended target.
The performance of antibodies can be affected by improper storage. It’s important to follow storage instructions on the datasheet provided by the manufacturer. For instance, at NeoBiotechnologies, we recommend aliquoting antibodies into smaller volumes sufficient for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70 °C) to avoid repeated freeze-thaw cycles which can degrade the antibody.
Inadequate tissue fixation or overfixation can result in poor staining. Adjusting the fixation time or trying a different fixative might improve the results. In cases of overfixation, the duration of immersion or post-fixation steps can be reduced. If immersion fixation can’t be avoided (for example, collection of postmortem tissues or biopsies in a pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents.
Incompatibility between the secondary and primary antibodies can also lead to problems. It’s important to use a secondary antibody that can interact with the primary antibody. For example, if the primary antibody was raised in rabbits, an anti-rabbit secondary antibody should be used.
Finally, the order in which reagents are used can impact the results. Make sure that the correct reagents are used and that they are added in the correct order. For example, if quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody.
In conclusion, successful use of monoclonal antibodies requires careful attention to many factors, from antigen presence to proper fixation and antibody compatibility. Armed with this monoclonal antibodies troubleshooting guide, you’re well-equipped to address any issues that may arise in your research.
When you partner with us at NeoBiotechnologies, you benefit from our decades of experience in the biotech industry. We take pride in our wide range of highly validated, monospecific Rabbit Recombinant Monoclonal Antibodies ideal for various applications. Our team of scientists is always ready to assist you in troubleshooting any issues you may encounter in your work with monoclonal antibodies.
The advent of monoclonal antibodies has revolutionized the field of biological research and biotechnology. Thanks to their high specificity and sensitivity, monoclonal antibodies have found application in a wide range of research fields, including immunohistochemistry, flow cytometry, Western blotting, and immunofluorescence.
As we look ahead, the role of monoclonal antibodies in future research is expected to keep expanding. The continued development and refinement of monoclonal antibody technologies, such as those manufactured by us here at NeoBiotechnologies, will undoubtedly play a significant part in this growth. Our extensive portfolio of over 10,000+ monospecific, monoclonal antibodies, both recombinant and hybridoma, are developed, manufactured, and rigorously validated by our team of scientists. They will continue to serve as indispensable tools for researchers around the world.
While monoclonal antibodies bring immense benefits to the field of research, they also come with their set of challenges. This is where the essence of our monoclonal antibodies troubleshooting guide comes into play.
Issues such as non-specific staining, high antibody concentration, and background staining in immunohistochemistry can potentially compromise the integrity of your experimental results. Therefore, proper troubleshooting is crucial to ensure the reliability of your work and to derive meaningful and accurate conclusions from your experiments.
We, at NeoBiotechnologies, understand the importance of proper troubleshooting in monoclonal antibodies usage. Our team of experts is committed to providing the necessary guidance and support to help you navigate through any issues you may encounter. So whether you are a seasoned researcher or a new entrant in the field, we are here to assist you in your journey.
In conclusion, monoclonal antibodies are an invaluable asset in the field of research, and troubleshooting is a crucial aspect of their effective usage. As we forge ahead, we at NeoBiotechnologies will continue to provide high-quality monoclonal antibodies and offer comprehensive support to researchers around the globe.
For more information on our products and services, explore our resources or contact us directly. We look forward to aiding you in your research endeavors.