I'm planning on doing a Western Blot to compare expression levels of recombinant proteins in E. coli, and I'm trying to find a suitable housekeeping protein to normalize total protein load in each lane. My samples are whole cell lysates - no purification steps.
I've come across papers where GAPDH and RecA have been used as housekeeping proteins in bacterial samples, but I've found just as many papers which state that these are unreliable. Are there other housekeeping proteins in E. coli that might be better choices? Or would I be better off just doing a Ponceau stain of my blot instead?
Any advice would be greatly appreciated! Thank you!
GAPDH is good for loading control. Many of the papers (but not all) that say GAPDH is not reliable tend to be industry papers trying to sell a newer technique to make money. While it could be under some circumstances there are GAPDH alternatives, stick with GAPDH for now.
Hi, it is always good to make a Ponceau stain to control Protein Transfer and it is also useful for normalization. But if you want to quantify something ( and not only estimate), perform an ELISA instead.
Dear Jacob Mack,
I have to disagree. Yes I do work for a company but in the last years a high number of papers have been published by research groups who are not cooperating with companies to sell things and they could clearly show, that Actin, GAPDH and all these proteins are regulated. In my time as a researcher in tumor biology I could see for myself how difficult it is to find a "house keeper". Actin was strongly regulated as well as GAPDH and Tubulin. To see your actual target you mostly need protein amounts that are way higher as the amounts you would load to see a "house keeper". They are so high abundant that you are saturating the membrane. With this you are not in a linear range anymore. Most groups do not even titrate the used antibodies. They are happy when the signal is strong and looks evenly. In the end you assume too many things that influence your results. Using e.g. a multiplex fluorescence approach to make the total protein visible alongside your target in a large linear range can improve the validity of your results. To use the total protein as normalization factor for a target was the original idea for Western blot. It wasn't possible then but now it is and it is very easy. The house keeper have always been a crutch and a well known bad one. It was a compromise you no longer need. So why not use the improvements of our time? It even saves time not to use a second antibody for a second protein detection.
Only my oppinion :)
You can use total protein normalization instead of looking for housekeeping proteins other than GAPDH. For better confirmation, perform two separate blotting experiments, one with total protain normalization and second with endogenous (house keeping) protein normalization.
It is acceptable to stain the membrane with Ponceau S after you have probed with antibodies directed against your protein of interest.
Regards,
John- Patrick
@ Christian P. Moritz : Interesting and informative paper. Many thanks.
John- Patrick
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