I am seeing upregulation of actin, GAPDH and tubulin by TGF beta treatment in cardiac and lung fibroblasts....is that true? When I checked the literature, many reports have suggested that TGF beta effects expression of these proteins but simultaneously I can see bulks of papers which have used these proteins as a loading control after TGF treatment....I am not able to understand how come this variability is coming up? Has anyone used any other loading control which does not fluctuate by TGF beta treatment?
Are you confident about your experimental design to answer TGFb1 induced phenotypes? Please contend with the idea that TFGb1 or for that matter any signaling molecule when used as a treatment should be done on quiescent cells i.e. serum starved .
Next when you set up your cell seeding into individual wells or dishes make sure you use a micropippetor to maintain a uniform seeding density. Make sure to have your seeding cell suspension stock vortexed before every seeding.
Do not add TGFb1 in micro liter volumes to individual dishes, prepare your treatment in media without serum and then use this media for experimentation. Im assuming youd treat for atleast 24 hours. For cardiac fibroblasts and lung fibroblasts use high glucose media with TGFb1.
Make sure your Bradford assay or MicroBCA method is giving consistent data, perform each read in triplicates.
For loading control use b-actin, gamma tubulin or RPL32 on your western.
Are you loading based on cell number in the assay? Especially if these cell numbers are from start of assay??
I routinely use TOTAL PROTEIN from lysate in Western. Use equal protein per lane. Then check for loading controls like b-actin, tubulin, histone, etc. Only then you can be confident that this is a direct protein-ONLY effect.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology