6 replies to this topic

[biochemical mayhem]

Recently, I found my protein to be expressed differentially during the life stages of Dictyostelium. I did this through western blot technique I am sure my antibody is working well. On the other hand, I performed cDNA amplication of my protein. This showed equal expression of mRNA at all stages of dictyostelium.
Can anybody make me clear why this difference occurs at the protein and mRNA level? I guess,  both the methods should show similar expression.

[PhDinAcronyms]

It could be that there is a change in something regulating the protein level. For example perhaps your protein is either being degraded at a more rapid rate or is no longer being degraded etc. There doesn't always have to be a change in mRNA levels for there to be a change in protein levels. Just depends on where regulation is occuring.

[biochemical mayhem]

thanks

[Tabaluga]

biochemical mayhem, on 09 April 2012 - 12:43 PM, said:

Recently, I found my protein to be expressed differentially during the life stages of Dictyostelium. I did this through western blot technique I am sure my antibody is working well. On the other hand, I performed cDNA amplication of my protein. This showed equal expression of mRNA at all stages of dictyostelium.
Can anybody make me clear why this difference occurs at the protein and mRNA level? I guess,  both the methods should show similar expression.

I fully agree with the post by PhDinAcronyms, and here's a suggestion: I don't know how long the life stages of Dictyostelium are, but could you measure mRNA and protein levels at different time points per life stage ? Maybe this would shed some light on whether there is really no early change in mRNA expression. Bear in mind the half-life of your protein. If mRNA levels are truly equal, it looks indeed like the regulation is not on transcriptional level.

[biochemical mayhem]

Tabaluga, on 10 April 2012 - 12:54 PM, said:

biochemical mayhem, on 09 April 2012 - 12:43 PM, said:

Recently, I found my protein to be expressed differentially during the life stages of Dictyostelium. I did this through western blot technique I am sure my antibody is working well. On the other hand, I performed cDNA amplication of my protein. This showed equal expression of mRNA at all stages of dictyostelium.
Can anybody make me clear why this difference occurs at the protein and mRNA level? I guess,  both the methods should show similar expression.

I fully agree with the post by PhDinAcronyms, and here's a suggestion: I don't know how long the life stages of Dictyostelium are, but could you measure mRNA and protein levels at different time points per life stage ? Maybe this would shed some light on whether there is really no early change in mRNA expression. Bear in mind the half-life of your protein. If mRNA levels are truly equal, it looks indeed like the regulation is not on transcriptional level.

Your idea is nice Tabaluga. I have tested the protein levels at different time points per life stages, but not the mRNA levels. Hope I will get some insights. To be sure I have to know about the half life of my protiens as well. Can you make me clear how we can find out the turnover rate of my protein?

[Tabaluga]

biochemical mayhem, on 11 April 2012 - 12:45 AM, said:

Your idea is nice Tabaluga. I have tested the protein levels at different time points per life stages, but not the mRNA levels. Hope I will get some insights. To be sure I have to know about the half life of my protiens as well. Can you make me clear how we can find out the turnover rate of my protein?

I haven't got any experience on measuring protein half-lifes, but these threads might help you:

http://www.protocol-...posts/7613.html
http://www.protocol-...osts/14678.html

Here's another possibly useful link:

http://www.springerl...page=2&locus=27

Edited by Tabaluga, 11 April 2012 - 11:31 AM.

[biochemical mayhem]

Tabaluga, on 11 April 2012 - 11:31 AM, said:

biochemical mayhem, on 11 April 2012 - 12:45 AM, said:

Your idea is nice Tabaluga. I have tested the protein levels at different time points per life stages, but not the mRNA levels. Hope I will get some insights. To be sure I have to know about the half life of my protiens as well. Can you make me clear how we can find out the turnover rate of my protein?

I haven't got any experience on measuring protein half-lifes, but these threads might help you:

http://www.protocol-...posts/7613.html
http://www.protocol-...osts/14678.html

Here's another possibly useful link:

http://www.springerl...page=2&locus=27

Thanks Tabaluga for providing some insights...I will have a look over them...