I repeatedly see these bands (see attachment) by coomassie staining of 5% SDS/PAGE gels that I run at 80V during 6 hours to detect a high-MW protein by Western blot. I use proteins from human fibroblasts as a source material.
I am currently struggling to find an internal loading control protein in the range between 150 and 300 kDa (proteins below this range run off the gel). As you can see in the image, my fibroblasts do express abundantly a protein of around 200 kDa and another one around 260 kDa... I was wondering if someone could guess the identity of these proteins to test them as loading controls. Any educated guess is welcome! :)
I realize I could send these bands to mass spec, but I don't want to expend resources in this procedure (for now) just to find a normalization protein.
Alternatively, if someone can recommend a suitable protein for normalization of Western blot in the MW indicated (150-300 kDa), I would also appreciate it very much. I am also currently looking into normalization of my Western blots by total protein staining, but I still would like to have an internal protein control for comparison.
Thank you in advance for your help.
Hi,
Why don't you use gradient gel to obtain more protein less than 117 kDa? If you can do this you can use calnexin (90 kDa) for normalization.
Or you can just add a certain pure protein which you know its MW into your lysate.
Hi,
You can use ProSieve™ QuadColor™ Protein Marker, 4.6-300 kDa from Lonza.
http://www.lonza.com/products-services/bio-research/electrophoresis-of-nucleic-acids-and-proteins/protein-electrophoresis/protein-stains-markers/prosieve-protein-colored-and-unstained-markers.aspx
I think you can run your same set of samples in another gel to get actin/GAPDH/ Histone H3 as loading control, it is acceptable.
Thank you for your suggestions, I will try them.
I would still be interested to know if there is any reliable protein for normalization in the high-MW range, for comparison with these methods... Perhaps not, since I can only find in literature methods in line with your suggestions, or using total protein normalization by protein staining of the membrane (something that I am also going to test).
Anchel, I am a little confused about your question, are you looking for a protein standards in the 150 Kdal to 300 Kdal range? If so both BioRad and Sigma-Aldrich offer standardized proteins for control. If you are attempting to identify or characterize these high molecular weight proteins it is a different story. First running you gel for so long is a bad idea. Either use a gradient gel or a higher percent acrylamide gel. For the best result you want your protein of interest to run in the middle of the gel. That is where the highest definition will be. But that may be difficult with big proteins. I would suggest to you that the proteins are most likely mucins of some sort or highly cross-linked proteins. I would start by running the samples with and without beta mercap both heat and unheated. I would also add a carbohydrate stain to protocol. These types of studies will help with a basic understanding of what the properties of the protein are.
Good Luck!
I would recommend blotting for talin-1(8d4 monoclonal from Sigma, about 220kDa) or a myosin heavy chain (220-260 kDa range).
Thank you for your answers. What I am looking for is a proper internal control for normalization in the high-MW range (not protein standards). Knowing the identity of those bands would help me to select candidates, because the expression is obviously good enough. I appreciate very much your suggestions (mucins, talin-1, myosin...), I will order antibodies and test them in some of my blots. Thank you very much!
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