I am trying to normalize a 100kDa protein from a membrane fraction taken from brain. Almost all the membrane housekeeping proteins (N-cadherins, integrins, Na/K ATPases) are nearly 100kDa, meaning I can't use them for within-lane normalization. Does anyone know of other reliable housekeeping membrane proteins that are substantially smaller or larger than 100kDa? References would be useful.
Also, I've seen in some papers that people normalize membrane proteins to tubulin (because it remains anchored to membranes?) but this seems invalid to me because contamination of cytosolic protein would weigh in more heavily on your result.
I'd also be curious to hear people's input about total protein normalization, as this could be a useful method for people who cannot find a stable housekeeping protein (loading control) that is the right size (i.e., doesn't overlap with your protein of interest)
There are specialized microdomains within the plasma membrane termed caveolae, which have coat presents that are ubiquitously expressed. Generally most tissues express caveolar coat proteins. Depending on your tissue, you can try Caveolin-1, Caveolin-3, Cavin-1 and Popdc1 (aka Bves). They are
You might have use caveolin (around 20 kDa), Flotillin (50 kDa) or LRP6 (130).
I am not sure about the kDa of these proteins but ı know that many group use these Ab for fractionation check and loading control.
Best,
Yunus
The last lines of evidence recommended to normalize WB againt the total protein load since signals obtained by loading controls could be saturated. There are several articles dealing with this topic or have a look at BioRad bulletins /ChemiDoc imagine system/.
Good luck
Maria
I can only agree with Maria. Before you fractionate, just adjust the volume of your lysis buffer to the weight of the brain, to have equal concentrations of your homogenates. Quick and reliable.
Cheers,
Lech
Related to Maria's response, does anyone have a preference for a particular total protein normalization method? I see some papers use ponceau and others (like the one in this link) claim that fluorescence is superior.
Article Total Protein Analysis as a Reliable Loading Control for Qua...
Hi Steve,
look at the attach, it may be helpful for you to decide. What kind of total protein staining methods you will use finally? It will also be the matter of your budget as well as a suitable equipment, I think :)
Best regards
Maria
The table Maria provided on total protein normalization was taken from this Biorad website.
http://www.bio-rad.com/en-uk/applications-technologies/total-protein-detection#2
They reference a few other useful papers (keep in mind they want to sell their fluorescence stainfree system but it does seem like sound method)
Andrea, I do not have fluorescence compatibility at the moment, so I am going with the cheap method (stain for total protein with ponceau, image, quantify total protein, then immunolabel for the protein of interest, etc). I'm also going compare the total protein normalization method to ones using various housekeeping proteins.
I'd be interested to hear people's advice on using ponceau for total protein normalization.
http://www.bio-rad.com/en-uk/applications-technologies/total-protein-detection#2
Hi,
in attach you can find some literature regarding total protein normalization. To housekeeping proteins: since I work with mitochondria I have often used mitochondrial COX 4 (cytochrome c oxidase, subunit 4) as a loading control. But in some experiments with different pathological conditions I noticed the change in signal intensity. I thought firstly, that there is a problem with loading or transfer. However after checking I found that there was no problem with those. So I tried total protein staining. Personally, I was unsatisfied with Ponceau staining for total protein normalization. I was unable to keep reproducibility, so I gave up this way of staining. Though I have stain-free equipment, I don't use this way due to increased costs and less variability to prepare desired gels suitable for my experiments. So I switched to Coomassie staining despite its disadvantages and lower sensitivity. Firstly I perform all immunodetections and then continue with staining. I always keep precisely the same period of staining and destaining and it works for now, with both PVDF and nitrocellulose membranes.Whether this way is OK and acceptable, I will see after recieving the reviewer's comments :). I am also curious to hear people's experience.
regards,
Maria
- Badges
- Science method