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Problems with stable transfection


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3 replies to this topic

#1 yean_ny_nie

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Posted 28 July 2009 - 08:13 PM

Hi,

We currently doing a stable transfection for the cell lines SH-SY5Y (human neuroblastoma) and also N2a (mouse neuroblastoma). We cotransfect the cells with pBabe-Hyg which contains the mammalian selective marker (hygromycin resistance) and pCMV-Sport6 with insert alpha synuclein.

We perform the transfection using electroporator ECM830 from BTX Application. We linearized the plasmid with EcoRI for pBabe-Hyg and SalI for pCMV-Sport6 and use 15ug of each plasmid. Below is the parameter:
Voltage: 230V
Pulse length: 8msec
Electrode type: 4mm gap disposable cuvettes

After the electroporation, we culture the electroporated cells back in growth medium. After 48hrs, we select the cells with hygromycin for 10days. For SH-SY5Y cells, we use 150ug/ml hygromycin and for N2a is 200ug/ml.

The cells grow into colony as expected and we harvest the cells to confirm their expression level of alpha synuclein. However, our western blot didnt show any overexpression of the desired protein.

We are wondering where goes wrong. We have tried using the same parameter for a prion protein insert with the same plasmid, and it is succesfully transfected. However, for alpha synuclein, it does not. Anyone have any idea regarding this?

#2 dxm

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Posted 25 August 2009 - 08:40 PM

1. Include a GFP vector control group, and check the GFP transfection efficiency before you collect.
2. You can try Fugene for transfection, it works quite good, but also quite expensive... :)

#3 bob1

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Posted 26 August 2009 - 04:33 PM

Is the plasmid correctly constructed - insert in frame?
Does the vector need to be induced for it to work?
Does your antibody work?
Have you looked for RNA expression?

#4 mrogan

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Posted 13 September 2009 - 07:20 AM

Hi - you may want to try posting this query also at www.pdonlineresearch.org, which is a new free community of Parkinson's disease researchers. your post would fit in the 'protocol Q&A' section:
PD Online Research Protocol Q&A

there are already some alpha synuclein related posts there.


Hi,

We currently doing a stable transfection for the cell lines SH-SY5Y (human neuroblastoma) and also N2a (mouse neuroblastoma). We cotransfect the cells with pBabe-Hyg which contains the mammalian selective marker (hygromycin resistance) and pCMV-Sport6 with insert alpha synuclein.

We perform the transfection using electroporator ECM830 from BTX Application. We linearized the plasmid with EcoRI for pBabe-Hyg and SalI for pCMV-Sport6 and use 15ug of each plasmid. Below is the parameter:
Voltage: 230V
Pulse length: 8msec
Electrode type: 4mm gap disposable cuvettes

After the electroporation, we culture the electroporated cells back in growth medium. After 48hrs, we select the cells with hygromycin for 10days. For SH-SY5Y cells, we use 150ug/ml hygromycin and for N2a is 200ug/ml.

The cells grow into colony as expected and we harvest the cells to confirm their expression level of alpha synuclein. However, our western blot didnt show any overexpression of the desired protein.

We are wondering where goes wrong. We have tried using the same parameter for a prion protein insert with the same plasmid, and it is succesfully transfected. However, for alpha synuclein, it does not. Anyone have any idea regarding this?






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