Hello all,
I am probing for the difference in expression levels of certain proteins involved in fatty acid transport and muscle remodelling in avian skeletal muscle homogenates. I tried using GAPDH as the housekeeping control. However, I found some variation in GAPDH expression levels within samples belonging to different treatment groups - subjected to different cold acclimatisation regimes.
Furthermore, I became aware of the fact that GAPDH expression levels, and perhaps those of other housekeeping genes, are not always constant under certain circumstances, and that total protein quantification is superior or even recommended by some journals.
I am currently using Ponceau S to check for post-transfer efficiency in 0.2µm nitrocellulose membranes. The Ponceau S staining seems to produce bands of even colour intensities between sample lanes loaded with "identical" protein loads (6-10µg).
However, I came across certain posts which suggested that Ponceau S has poor sensitivity, and hence I am thinking about using SYPRO ruby stain, which is supposed to have markedly higher sensitivity and dynamic range.
I saw some recommendations for Pierce MemCode in this forum, but they don't seem to be manufactured anymore. Other alternatives suggested include Invitrogen No-Stain™ Protein Labeling Reagent, Biotium One-Step Lumitein™ & VersaBlot™, LI-COR REVERT™, Bio-Rad Stain-Free™ and Amersham™ Quickstain, etc.
I thus have the following questions -
1. Have any of you fellow researchers had success in performing total protein quantification using SYPRO ruby stain FOLLOWED by immunoblotting for target proteins?
2. What specific protocol did you use if the answer to question 1, is "yes"
3. If the answer to question 1 is "no", what alternative would you recommend?
4. If any of you had success with a total protein quantification stain which was better than SYPRO ruby, specifically in terms of simpler/faster protocol and/or no toxic chemical disposal requirements, please suggest the stain and it's corresponding protocol.
Thanks and regards
Sampath
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