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30 November, 2023 by Anshul (neobio)
The success of a biological experiment hinges heavily on the specific binding of antibodies to their target proteins. But improper target recognition due to nonspecific antibody binding usually leads to skewed results or wrong interpretations. Our expert, Dr. Atul K. Tandon, CEO of NeoBiotechnologies, concurs on the crucial nature of antibody validation using Western blot technique, to ensure the specificity of our primary antibodies. This necessity for accuracy presents a daunting challenge to researchers and scientists like yourself who strive for precise, reproducible results in applications like Immunohistochemistry, Flow Cytometry, and Western Blotting.
Antibody validation implies a meticulous confirmation that an antibody is reliably detecting its intended protein target in a specific application. A properly validated antibody ensures the reproducibility of your results, reducing the risk of inaccuracies and saving both time and resources in your research journey.
Western Blot is an indispensable tool in antibody validation, offering the resolving power of electrophoresis, combined with the specificity of antibodies and sensitivity of enzyme assays. This technique allows you to see if the antibody can bind to the targeted protein and creates a visible band on a gel that corresponds to the protein of interest. Although it sounds simple, it’s not uncommon to encounter additional, non-specific bands that can crowd and distort quantification.
While accurate validation is indispensable, it doesn’t have to cause a major dent in your budget. Cost-effective validation relies on precise optimization of primary antibody concentrations, judicious choice of buffers based on the experiment, and leveraging affordable yet reliable antibodies. Fortunately, at NeoBiotechnologies, we offer an extensive portfolio of over 10,000+ monoclonal antibodies, rigorously validated by our team of scientists, that marry affordability with precision.
Here’s an informative infographic detailing the process and importance of antibody validation:
The Western Blot (WB) technique is one of the most reliable ways to validate an antibody. It’s a multi-step process that involves preparing lysates from cells or tissues, running the blot, confirming antibody specificity, and using a densitometer and imaging software for objective measurement.
Before you start the validation process, you need to prepare lysates from cells or tissues that express the protein of interest. This step is crucial as it helps identify the right lysates to use for the western blot. At NeoBiotechnologies, we have a team of experienced scientists who can assist you with this task, ensuring the proper preparation of lysates.
Once the lysates are prepared, you’ll run the western blot. This involves transferring proteins from a gel onto a membrane, which is then probed with a primary antibody specific to the protein of interest. The band size is then checked for the expected molecular weight. The expected appearance of a band at the correct molecular weight is a strong initial indicator of a valid antibody.
It’s not enough for the antibody to bind to the protein – it needs to bind specifically and with low cross-reactivity to other proteins. To confirm antibody specificity, you can probe identical samples with multiple antibodies in parallel. This gives a quick visual indication of antibody specificity, essential for the validation process.
To make a permanent record of the signal generated on a Western blot, a densitometer is used to scan the blot or film. Imaging software, such as the one we use at NeoBiotechnologies, is then used to compare the signal generated by the bands detected on the Western blot. This objective measurement is crucial in confirming the results of the western blot.
Remember, antibody validation using western blot is not a one-size-fits-all approach. Depending on your specific needs and circumstances, you might need to adjust some steps. But with the right approach and resources, you can perform antibody validation western blot on a budget without sacrificing the quality of the results.
In the next section, we’ll discuss some advanced techniques for enhanced antibody validation. Stay tuned!
After the basic steps of antibody validation western blot, you may opt to employ advanced techniques for enhanced validation. These methods provide a more comprehensive assessment of antibody specificity and can give you a higher degree of confidence in your results. Here are some advanced techniques you can employ:
Orthogonal validation is an effective way to cross-verify the results obtained from Western blotting. This approach involves comparing the protein expression data from the Western blot with RNA-Seq data. If the patterns of protein and RNA expression align, this provides strong support for the validity of the antibody. However, it’s important to keep in mind that there might be post-transcriptional regulations affecting the correlation between RNA and protein levels.
This technique involves using other independent antibody-based assays such as ELISA, immunohistochemistry, or tissue microarrays to cross-validate the results of your Western blot. For instance, if the staining pattern observed in an immunohistochemistry experiment matches the Western blot result, it provides additional evidence of the specificity of the antibody .
Genetic validation is a robust method to confirm the specificity of an antibody. It involves using techniques like RNA interference (RNAi) or CRISPR-Cas9 to reduce or eliminate the expression of the target protein, and then observing whether the signal detected by the antibody also decreases or disappears. This provides strong evidence that the antibody is indeed specific to the protein of interest.
This method involves overexpressing or tagging the protein of interest in a sample, and then testing whether the antibody can detect the increased presence of the protein. If the antibody can detect the overexpressed or tagged protein, this confirms that it is capable of binding specifically to the target protein. However, be cautious with this method, as overexpressed proteins can sometimes cause off-target binding .
This innovative technique involves comparing the staining pattern and protein size detected by the antibody with results obtained by capture Mass Spectrometry (MS). If the size detected by the antibody matches the size detected in migration capture MS, it confirms the specificity of the antibody.
At NeoBiotechnologies, we understand the importance of antibody validation for the success of your research. That’s why our team, led by Dr. Atul K. Tandon, rigorously validates all our monospecific Rabbit Recombinant Monoclonal Antibodies using these advanced techniques. Our commitment to quality ensures that you have access to reliable antibodies that will yield consistent and accurate results.
While antibody validation is a crucial step in ensuring the reliability and accuracy of your experimental results, it doesn’t need to be a financially draining process. By making smart choices about the antibodies you use, streamlining your protocols, and selecting the right buffers, you can conduct reliable antibody validation using Western blot without breaking the bank.
Choosing the right antibodies is a critical first step in your validation process. At NeoBiotechnologies, under the guidance of our founder and CEO, Dr. Atul K. Tandon, we manufacture over 500 highly validated, monospecific Rabbit Recombinant Monoclonal Antibodies. Known for their precision and consistency, these antibodies are ideal for applications such as Immunohistochemistry, Flow Cytometry, Western Blotting, or Immunofluorescence. By using these cost-effective antibodies, you can trust that you’re investing in quality without straining your budget.
The second key to affordable antibody validation lies in optimizing your protocols. As every researcher knows, time is money, and inefficient protocols can lead to wasted reagents and costly repetitions of experiments. For example, the incubation period for an antibody can vary from a minimum of one hour to overnight at 4°C. You need to determine the optimal incubation period for each antibody to avoid sensitivity issues or background staining. Our antibody protocols and troubleshooting tip guide can help you fine-tune your protocols to achieve the best results efficiently and affordably.
Finally, selecting the right buffers for your experiment can significantly impact the performance of your antibody validation Western blot. The majority of antibody assays use two buffer types: PBS or TBS. Factors like pH can influence buffer performance. Therefore, it’s important to determine the optimal buffer for your experiment. Through careful selection and preparation of your buffers, you can ensure that your validation process runs smoothly and cost-effectively.
In conclusion, with the right choices and optimizations, validating your antibodies can be both affordable and reliable. Here at NeoBiotechnologies, we’re committed to providing you with high-quality, cost-effective antibodies and expert guidance to help you achieve your research goals.
The process of antibody validation using western blot is not just a procedure, but a vital step that ensures the accuracy and reproducibility of your research. The success of your experiments largely depends on the specificity and selectivity of your antibodies. Therefore, adopting rigorous validation processes is crucial. As highlighted by the International Working Group for Antibody Validation (IWGAV) and our expert, Dr. Atul K. Tandon, evaluating antibody specificity and reproducibility is a minimum requirement in this process.
However, antibody validation should not just stop at this minimum requirement. The use of advanced techniques such as orthogonal validation, independent antibody validation, genetic validation, recombinant expression validation, and migration capture MS validation can provide a more comprehensive and reliable assessment of your antibodies. These techniques serve as layers of confirmation that ensure the antibodies you’re using are indeed specific to your targets and can produce reproducible results.
Here at NeoBiotechnologies, we understand that research costs can be a significant constraint. However, we believe that budget constraints should not compromise the quality and reliability of your research.
We are proud to provide over 500 highly validated, monospecific Rabbit Recombinant Monoclonal Antibodies, which are ideal for various applications such as Immunohistochemistry, Flow Cytometry, Western Blotting, and Immunofluorescence. Our antibodies are rigorously validated by our team of scientists, ensuring that they are of high quality and can deliver reliable results.
We also provide support in optimizing your antibody validation protocols to reduce costs without compromising the quality of results. We guide researchers on how to choose the right buffers based on experiment requirements and how to optimize the amount of sample loaded, western blotting conditions, and detection/visualization methods to ensure cost-effectiveness.
In conclusion, comprehensive antibody validation is a vital aspect of research that ensures the accuracy and reproducibility of results. With NeoBiotechnologies, you can achieve reliable results on a budget. Our high-quality, cost-effective antibodies, coupled with our expert guidance on protocol optimization, can help you achieve your research goals without breaking the bank.
For more information about our Rabbit Recombinant Monoclonal Antibodies, don’t hesitate to contact us. Our team, led by Dr. Atul K. Tandon, is always ready to assist you with your research needs.
