Protamine Sulfate Protocol for Transfection Enhancement

In Brief Download the PDF of this protocol

Step 1: Prepare DNA Plasmid

Sufficient plasmid DNA must first be generated. Start to amplify the quantity of plasmid by performing bacterial transformation. Grow the transformed bacterial culture overnight (in a 37°C shaker) and extract the plasmid DNA using a DNA MAXIprep kit.

Step 2: Prepare HEK Cells

Culture HEK293T cells in HEK media (DMEM/F-12 media, FBS, non-essential amino acids, L-glutamine) within humidified incubators under standard conditions (37˚C, 5% CO₂, and 21% O₂). Once the culture has reached high confluency (>90%), the cells are ready for transfection.

Step 3: Prepare Transfection Media

Immediately before transfection, replace the HEK culture media with DMEM.
Prepare the transfection media, which consists of an optimized ratio of DMEM, DNA plasmid, lipofection reagent, and protamine sulfate stock solution (see Table 1).
Typically, protamine sulfate is used at a concentration of 5–10 µg/mL (see Table 2).

Step 4: Transfection

Gently mix the transfection media before adding dropwise to the culture plate to prevent damaging the cells.
Place the cells immediately in the incubator under the following conditions overnight: 37˚C, 5% CO₂, and 21% O₂.

Step 5: Harvesting

Return the culture to a standard maintenance incubator. Leave the cells for 48–72 hours post-transfection before proceeding to downstream applications (e.g., harvesting virus from media).

Table 1: Protamine Sulfate Stock Solution Preparation

Protamine sulfate (Cat. No. 8822) is supplied as a 10 mg solid and is soluble in water.
To generate a working solution, protamine sulfate should first be dissolved in 1 mL sterile water to create a 10 mg/mL stock solution.

Stock Solution Protamine Concentration (mg/mL)	Stock Volume (mL)	Protamine Sulfate (solid, mg)	Sterile Ultra-Pure Water (mL)
10	1	10	1

For more information on preparing stock solutions, see our Molarity Calculator.

Table 2: Protamine Sulfate Working Solution Preparation

Protamine sulfate working solution is generated from the 10 mg/mL stock solution. This stock solution can be further diluted in culture media to generate the desired working concentration for transfection or transduction. Example of dilutions are shown below:

Final Desired Working Concentration (µg/mL)	Dilution Factor	Stock Solution Protamine Concentration (mg/mL)	Volume of Stock Solution Protamine (µL)	Volume of Culture Media (mL)
5	1:2000	10	25	50
6	3:5000	10	30	50
8	1:1250	10	40	50
10	1:1000	10	50	50

For more information on calculating dilutions, see our Dilution Calculator.