Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device
Transcript
To begin, prepare a 100 kilodalton molecular weight cutoff centrifugal filter by adding the full volume capacity of PBS. Centrifuge for five minutes at 2,800 times g at 4 degrees Celsius. Discard the flow through, and any remaining PBS, and fill the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 times g at 4 degrees Celsius until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary until the entire sample has been reduced sufficiently.
Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described before and repeat this step five times to ensure complete washing and buffer exchange. Then, recover the 100 kilodalton-concentrated CFP retentate, or 100R, according to the ultrafiltration device specifications. Wash the filter with a minimal volume of 1x PBS at least three times, and pull the wash with the 100R to maximize the recovery.
