Dear Sir or MadamWe have troubles to reach a virus concentration...

protocol Protocol: Fluorometric Estimation of Viral Thermal Stability
Dear Sir or Madam

We have troubles to reach a virus concentration of 0,1 to 0,24 mg/mL. So our question is how you determined the protein concentration.

Yours sincerely

Kathrin Schlicht
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Vamseedhar Rayaprolu Author Answered Mar 5, 2016

Pacific Northwest Cryo-EM Center, Oregon Health and Sciences University, Portland, USA

Hi,

Sorry for the delayed response. Please look at the original reference (I posted the link in the previous post). Also look at references 51-54 in that link. This method was developed by our collaborators in SF9 using a baculoviral expression system. Let me know if you need more information.
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Kathrin Schlicht Answered Mar 2, 2016

Universität Bielefeld

Hi Vamseedhar,

we produce our AAVs by calcium phosphate-based transfection of plasmid DNA into HEK cells. The viral titer we reach is bellow the titer you need to get a protein concentration that high even after concentration by a molecular cut off filter. So we can`t perform DSF. We really like to test this method. Maybe you know how many HEK cells were transfected.

Thank you for your quick response.
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Vamseedhar Rayaprolu Author Answered Mar 2, 2016

Pacific Northwest Cryo-EM Center, Oregon Health and Sciences University, Portland, USA

Hi Kathrin,

We determined concentrations using gel densitometry. You could also use UV 280 to determine the protein concentration but because our virus sample also had DNA, we preferred running the gel and doing the densitometry. You can increase your protein concentration using molecular a weight cut off filter though AAV samples tend to stick to the filters quite a bit. The samples were obtained from our collaborators. For more information on purification of the virus and checking integrity, please refer to http://jvi.asm.org/content/87/24/13150.full

I hope this helps.
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