what should be used as controls during western blotting? - (Jun/24/2015 )

Hi guys,

i posted this question on molecular biology but nobody replied...may be it's not a serious question..Let's say i am doing western blot.

If i have an immortalized cells (using Htert) overexpressing Akt, what should be the control cell for comparison?

Q 1. Can it be like this?

control - immortalized cells expressing GFP construct

OE cells- immortalized cells overexressing protein of interest (Akt)

Q 2. or can the control be just immortalized cells as they already have Htert construct?

For downregulation, i know the control is generally a non-silencing construct.

But what do you use as a control against overexpressing cell ?

thanks a lottt

You would normally just use the same cells without the GOI being overexpressed.

bob1 on Wed Jun 24 21:08:17 2015 said:

You would normally just use the same cells without the GOI being overexpressed.

hi, thanks for the reply.

but isn't the same cells with overexpression of gfp a better control?  it will show that only the GOI overexpression leads to the consequences and not overexpression of any other protein (e.g gfp)

what do u say?

Depends on what you are looking at -you haven't given us any information about your experiment.

If, for instance, you were just looking to see whether Akt was overexpressed, you wouldn't need any controls other than a cell line that has normal expression. On the other hand, if you are looking at some other factor that might be influenced by the expression of Akt, then overexpression of another protein might be appropriate. I wouldn't use GFP for this purpose, overexpression of GFP can cause protein aggregation under some circumstances.

bob1 on Wed Jun 24 23:32:37 2015 said:

Depends on what you are looking at -you haven't given us any information about your experiment.

 

If, for instance, you were just looking to see whether Akt was overexpressed, you wouldn't need any controls other than a cell line that has normal expression. On the other hand, if you are looking at some other factor that might be influenced by the expression of Akt, then overexpression of another protein might be appropriate. I wouldn't use GFP for this purpose, overexpression of GFP can cause protein aggregation under some circumstances.

hmm.  well the idea is both. the overall experiment is to check how is the expression of klf4 in Akt-overexpressed cells. the hypothesis is Akt overexpression leads to enhanced klf4 expression.

SO, in the same blot, i would need to examine Akt -- to show that the overexpression has worked...

and then i also check the expression of KLF4-- to check whether its expression is higher than in control..

Essentially, my questions are- can the control in this situation be:

1. same cells without the GOI being overexpressed?

2. cells expressing some random plasmid which wouldn't affect akt n klf4. If not GFP, what are other suggestions if u have?

3. can the non-silencing control be used as control here as well?