Hi all,
I´m currently working on the recombinant expression of a human transmembrane protein in High Five insect cells by a baculovirus expression system and I would like to know if there is a good candidate for a loading control in Western-Blotting for these cells, i.e., any good housekeeping protein that would be fit for this purpose. Since my protein´s molecular weight is around 40 kDa, I discarded using classical options like beta-actin and GAPDH, but I´d like to know other alternatives and your user experience with them.
Thank you in advance for your help!
Dear Alfonso,
I would suggest tubulin or vinculin. However, you need to be sure that appropriate antibodies specific for your system are available. See the link for more details.
https://www.labome.com/method/Loading-Controls-for-Western-Blots.html
Another appropriate way is to quantify the protein in the lysates and load equal proteins in the gel, which normally works very well.
I hope this information is helpful.
Goodluck
Dear Ramachandramouli,
thanks for your reply. I always load the same amount of protein in the gel after protein quantification by BCA or Bradford Assay, but since I am using samples from different cell sources (insect cells and mammalian cells) and in order to compare them and do a Western Blot quantification, I am looking for a good loading control for my High Five cell samples.
I will take into consideration your recommendation of tubular/vinculin. Do you have any experience working with them or any good reference of their use in baculovirus expression systems (using Sf9 or High Five cells)?
Thanks a lot for your help.
Best,
Alfonso
If you would like to compare the expression between two different cell souces (and from different species), I doubt whether it is an appropriate way to use loading controls. I assume loading equal protein amounts should be fine.
However, I do not have experience using loading controls for baculovirus expression system as my question was too simple (whether my protein expressed well or not). If you really want to use a loading control, you need to dig the literature or the internet for a proper antibody.
Goodluck
Hi again, Ramachandramouli.
As for any other loading control, I am just looking for some kind of housekeeping protein that could help me to normalize the signals from my insect cell samples, since they correspond to different cultures infected with different amounts of virus and I want to quantify accurately the amount of recombinant protein produced by each culture.
Anyway, thank you very much for your recommendations. I will take them into consideration.
Best,
Alfonso
Amigo Alfonso.
Have a look to this paper. I hope it can help you.
Un abrazo
Mario
Hi, amigo Mario.
I already heard about this method, but your attached paper is really good. I will take it into consideration for my future WBs.
I hope you´re doing well (are you now in Oxford?). Un abrazo desde Alemania,
Alfonso
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