While the data sheet for the beta-actin antibody I use (see attached) suggests a dilution range of 1:100-1:1000, I've found that following this recommendation leaves me with bands so thick and bright that it makes comparison difficult and dodgy.
After lots of trial and error, I've found that a dilution of 1:17,500 gives me thinner, more reasonable bands, but it shocks me that I'd need to deviate so drastically from the manufacturer's recommendation. Has anyone else encountered a similar phenomenon? Or have any idea why this might be happening? If it helps, my protein usually comes from RAW264.7 or NIH3T3 cells.
Hi
it is highly conserved cytoskeletal protein (abundant). It is expressed in high levels in all cell types hence the name housekeeping.And also highly conserved among certain species* Thats why in many assays it serves as a good loading control. The MW appx 42kda. As for the band thickness and intensity let me send you a picture of my western blot actin and take a look.
Any extra infos from more experts would be also a great add in*
Callan Calloway Frye I am using the same antibody at a 1:5000 ratio. It works fine. The expression of actin depends on the cell line although from the same tissue. Hope it helps.
Hamadi Madhi, ill agree with what you said cause i ve followed the 1:1000 ratio.
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning