shRNA Titer by p24 Assay - ELISA for the p24 Viral Coat Protein

Protocol courtesy of Sigma MISSION^® RNAi Team

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• Introduction
• Materials
• Protocol
• Tips

   

Introduction

We determine the TU/ml based upon the p24 assay from ZeptoMetrix. The titer calculation is determined by using the conversion factor from Didier Trono to convert from concentration of p24 to viral titer. There are approximately 1 x 10⁴ physical particles of lentivirus for every pg of p24 antigen. Based on ELISA results, this works out to be 100 transducing units (TU) for every pg of p24 antigen present.

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Materials

• Lentivirus targeting your favorite gene
• p24 ELISA from ZeptoMetrix
• Calculator or Spreadsheet Program

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Protocol

Day 0

0.1		Dilute samples between 750 and 3000 fold.
0.2		Utilize the p24 ELISA kit from ZeptoMextrix and follow the manufacturer's instructions.
0.3		ALWAYS include blanks to “zero” the plate reader.
0.4		ALWAYS utilize a p24 standard curve for calibration.
0.5		For the standard curve, utilize two independent dilutions with five concentrations of p24 plus a concentration of 0.
0.6		Plot the standard curve as concentration of p24 vs. OD450
0.7		The fit in the standard curve should yield an R2 > 0.95.
0.8		Determine the slope m and the intercept b. (You may also utilize the Calibration Sheet in the attached spreadsheet)
0.9		Calculate TU/ml for your diluted virus based upon the following equation (You may also utilize the Data Sheet in the attached spreadsheet)

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Tips

Utilize the attached spreadsheet (2.09 Mb) for calculation of TU/ml.

Derivation of conversion factor:

a. (2 x 10³ molecules) x (24 x 10³ Da of p24 per PP) = (48 x 10⁶)

b. 48 x 10⁶/Avogadro’s constant = (48 x 10⁶) / (6 x 10²³) = 8x10^-17 g of p24 per PP

c. There is approximately 1 PP per 1 x 10^-16 g of p24

d. 1 x 10⁴ PP per pg of p24

e. 100 TU per 1000 PP

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