Hello, esteemed scholars and doctors,
I hope this message finds you well. I apologize for the interruption, but we are currently engaged in a project that involves assessing the oligomerization level of a protein. In our search on PubMed, we discovered that the crosslinker recommended for this protein is EGS (ethylene glycol bis(succinimidyl succinate)).
However, we find ourselves in need of guidance on how to effectively utilize this crosslinker. Based on existing research, cells were harvested by scraping, washed twice with PBS, and incubated with 0.5 mM EGS in PBS, pH 7.4 at 30 °C for 20 min. And then to quench excess crosslinker, 1.5M Tris HCl(pH 7.8) was added to a final concentration of 20 mM and incubated for 5 min at room temperature and the samples were centrifuged at 10,000×g for 5 min. Subsequently, the pellet was lysed in NP-40 lysis buffer via sonication on ice, followed by BCA and WB procedures.
I am seeking advice on the appropriate amount of EGS to add per 6-well plate or for 1*10^6 cells, as well as the recommended quantity of 1.5M Tris to use for quenching the EGS.
Additionally, I understand that NP-40 lysis buffer is chosen for its gentle nature. However, I am curious if it would be permissible to use a lysis buffer containing SDS, considering its compatibility with SDS-PAGE blotting.
Would it be necessary to perform high-temperature denaturation and include a loading buffer for SDS-PAGE electrophoresis with the final samples?
If you have an alternative or more efficient protocol to share, we would be immensely grateful for your insights and expertise.
Thank you for your time and assistance.
Best regard！
Hi,
I am not an expert in the field of cross-linking of proteins and its certain applications but I may help you to find some solutions for lysis and PAGE;
You can use SDS containing buffers like RIPA for cell lysis and it is compatible to use at SDS-PAGE analysis. In many cases RIPA buffer is effective as NP-40 alone and enzymatic and/or mechanical/physical assistance during cell lysis will definitely improve the yield...
For gel electrophoresis, in general, heat induced denaturation and Laemmnli buffer composition is used to form protein as completely anionic (SDS coating), denaturated (high heat), linear (reducing the S-S), slightly alkaline (Tris buffer), and partially viscous (glycerol additive)...Tracking dye (bromophenol B) is also added to follow the run...In these informations, you may exclude some of these ingredients and modify your electrophoresis assay...The behaviour of your protein and purpose of your analysis will direct you to choose the final composition...(Look for similar techniques that modifies the sample prep; e.g. BN-PAGE, Native-pAGE, Tricine-PAGE, urea-page...)..In some practices since membrane proteins are prone to be aggregated at high heat treatment, for SDS-PAGE analysis this part can be excluded...If the denaturation and results is not effective, you may modify the sample buffer by adding urea to increase denaturation if the heat is not applicable for your assay...
For the cross-linking the protocol you shared seem to me much more like to be a cross-linking strategy for secreted proteins or cell surface proteins. To characterize the oligomerization of your secreted protein, is it necessary to lyse the cells after quenching the EGS activation or if you aimed to crosslink the proteins other than extracellular matrix, why you treated the intact cells with EGS and removed after the application...Sorry if I cannot understand the purpose clearly...
Good luck,
İEA...
İsmail Emir Akyildiz Happy New Years，Dr.Akyildiz. I apologize for the delayed response. Your insights into lysis and PAGE techniques are truly profound and appreciated.
Considering our proteins are mitochondrial and membrane-bound, it seems, following your suggestion, that incorporating β-mercaptoethanol into the SDS-PAGE loading buffer might be beneficial.
Regarding the issue of protein cross-linking with EGS, I've learned that there are two types of reagents: EGS and Sulfo-EGS. The former can penetrate the cell membrane, while the latter cannot, making it suitable for surface protein cross-linking. Our recent acquisition of EGS has arrived, and I plan to implement the previous protocol to see if we can achieve the expected results.
Once again, I am grateful for your insights and sharing of knowledge.
Best wishes and a Happy New Year.
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