Hello all,
I am currently doing Dot Blots to confirm that my antibody is present after an Immunoprecipitant run and for some reason my signal is coming through in the FT and Wash but not in the Elution. My colleague and I are hypothesizing that the antibody we are testing for is stuck to the beads that we are using (AVIDIN for the 1st antibody and HRP for the 2nd) but we are not quite sure if this is the case or if something else is occurring. The kit we are using for Immunoprecipitating is a Thermo Pierce Classic IP kit. We don't know what else to do as we have been tweaking things like the pH for the Elution Buffer and we are kind of at a loss. The next thing we are going to try doing is modifying the wash agent by using 1x PBS but we aren't sure if that will solve anything. Does anyone have insight to this problem or perhaps a similar experience? Sorry this is my first time using ResearchGate so I hope my question comes across clearly.
I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?
Your issue with the antibody signal appearing in the flow-through (FT) and wash fractions but not in the elution suggests that the antibody might be adhering to the beads used in the immunoprecipitation (IP) process. This could be due to suboptimal elution conditions. To address this, first ensure that the pH and ionic strength of your elution buffer are well-suited for disrupting the specific antibody-bead interactions. Sometimes, adjusting to a more acidic pH or increasing the salt concentration can enhance elution efficiency[1]. Additionally, incorporating a gentle detergent in the elution buffer might help release bound antibodies more effectively[2].
Ensure that the duration and temperature of the elution step are sufficient to facilitate the detachment of the antibody from the beads[1]. If you are concerned about the compatibility of your elution buffer with downstream applications, verify that it does not interfere with detection methods, such as dot blots or other assays you plan to use. Also, consider the potential role of the specific binding characteristics of the beads (e.g., AVIDIN and HRP) and how they might affect antibody retention[2]. Experimenting with different wash agents, like 1x PBS as you mentioned, could also help in optimizing the elution efficiency to ensure that the antibody is not lost in the wash steps[1].
By systematically adjusting these parameters, you should be able to identify the optimal conditions for eluting your antibody effectively from the beads.
Reference
[1]
Kulyk, V., Duriagina, Z., Kostryzhev, A., Vasyliv, B., Vavrukh, V., & Marenych, O. (2022). The Effect of Yttria Content on Microstructure, Strength, and Fracture Behavior of Yttria-Stabilized Zirconia. Materials, 15.
[2]
Kulyk, V., Duriagina, Z., Vasyliv, B., Vavrukh, V., Kovbasiuk, T., Lyutyy, P., & Vira, V. (2022). The Effect of Sintering Temperature on the Phase Composition, Microstructure, and Mechanical Properties of Yttria-Stabilized Zirconia. Materials, 15.
Park Sowon
Thank you for your input. I will be in the lab this Monday so I will talk to my supervisor and see what the best course of action is. I will let you know as the week develops.
Thank you,
Brandon
Lisa Smith
Thanks Lisa! I don't see your message, is it supposed to be in my inbox?
So I tried using 1xPBS with our method and unfortunately it yielded unsatisfactory results. I will try modifying the procedures with the additional suggestions provided and see if anything is yielded next week.
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