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Determining phosphorylation status of a protein by transfection


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#1 Shamoon Saljuqi

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Posted 07 January 2014 - 04:15 PM

One part of my experiment was to analyse the phosphorylation status of Dvl2. I had transfected cell lines with Wnt6 and Dvl2 to see the effect wnt 6 would have on Dvl2. Following transfection protocols cells were starved for 24 hours and then harvested. I confirmed successful transfection of samples with Dvl2 and Wnt6 using western blot analysis. The conditions were as follows:

 

(Control)       (Dvl2)       (Wnt6)          (Dvl2 and Wnt6 co-transfection)

 

One problem is that Dvl2 expression in the conditions were not equal even though B-actin showed loading to be equal.

The expression of Dvl2 in the (Dvl2 + Wnt 6) were lower than in (Dvl2 alone). I don't understand this, does this mean that regardless of protein loading being equal Wnt 6 is downregulating expression of Dvl2 or that transfections worked better in (Wnt6) condition than in (Dvl2+Wnt 6).

 

I need help in analyzing these results. What do they mean?



#2 jerryshelly1

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Posted 07 January 2014 - 06:30 PM

Did you do a transient transfection or did you generate a stable cell line. If you generated a stable cell line, were you using vectors that had different selection markers?

 

I find that is it is always difficult to make an assumption like that in transfected cells. When you transfect cells, you are placing them under extreme stress that isn't always "correlation equals causation."

 

Does that make sense?



#3 bob1

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Posted 07 January 2014 - 07:13 PM

You need a few more controls here - you need to have a control that is an empty plasmid (preferably an empty version of the one wnt6 is in) co-transfected with dvl2 and the opposite -empty backbone of dvl2 cotransfected with wnt6.  I would also include co-transfection of off-target genes - a gene that is known not to affect dvl2 expression for instance.

 

It could easily be that the the expression is lower because it is a co-transfection so fewer copies of the dvl2 plasmid were getting in (or perhaps you used less plasmid in the first place and made up the balance of DNA amount with wnt6 plasmid?), or it could be that wnt6 expression affects the overall cellular production of protein, or alters cell cycle state which could be required for dvl2 expression (I don't know, as I don't work with either of these proteins - just throwing ideas out there).  Or it could be that you are adding twice as much DNA (and corresponding transfection reagent?) and the cells don't like this much.



#4 Shamoon Saljuqi

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Posted 07 January 2014 - 07:30 PM

I did a transient transfection. Yeah I understand what you mean. I just don't understand how to interpret this when I'm writing up my thesis. 

 

 The plan was this:

 

Transfect cells transiently

 

Confirm transfection by western blot analysis

 

If successful, immunoprecipitate samples for Dvl2 (FLAG-tagged). This would mean the only protein that should remain is Dvl2 in the (Dvl2) and (Dvl2+Wnt 6) transfected samples

 

Run western blot on immunoprecipitated samples, confirm presence of Dvl2 only in samples Tx with Dvl2.  Probe for phosphotyrosine.

 

 

This experiment was set up by my professor but something about it doesn't seem right.   

 

 

Did you do a transient transfection or did you generate a stable cell line. If you generated a stable cell line, were you using vectors that had different selection markers?

 

I find that is it is always difficult to make an assumption like that in transfected cells. When you transfect cells, you are placing them under extreme stress that isn't always "correlation equals causation."

 

Does that make sense?



#5 Shamoon Saljuqi

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Posted 07 January 2014 - 07:40 PM

You're right. There is way too many variables in this experiment I just don't understand why my professor didn't think of this. I am doing this for my thesis and it's too late to re-do the experiment. So many times I had thought of these problems but my professor said it's fine.... It just doesn't seem right

 

 

It could easily be that the the expression is lower because it is a co-transfection so fewer copies of the dvl2 plasmid were getting in (or perhaps you used less plasmid in the first place and made up the balance of DNA amount with wnt6 plasmid?), or it could be that wnt6 expression affects the overall cellular production of protein, or alters cell cycle state which could be required for dvl2 expression (I don't know, as I don't work with either of these proteins - just throwing ideas out there).  Or it could be that you are adding twice as much DNA (and corresponding transfection reagent?) and the cells don't like this much.



#6 bob1

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Posted 08 January 2014 - 11:43 AM

 

You're right. There is way too many variables in this experiment I just don't understand why my professor didn't think of this. I am doing this for my thesis and it's too late to re-do the experiment. So many times I had thought of these problems but my professor said it's fine.... It just doesn't seem right
 

These problems are all good things for the discussion section.  With regards to the IP - if you are looking for a direct interaction between Dvl2 and Wnt6 then you could do a look for the presence of wnt6 in your Dvl2 IPed samples.






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