Millipore (Amicon®) Ultra Centrifugal Filters/ micro-concentrators do not need any priming or prewash step for using for the first time. It comes ready to use and are not intended to be reused.
Amicon Stirred Ultrafiltration Cells are also used for protein concentration (RELATIVELY LARGER VOLUME), gas pressure is applied directly to ultrafiltration cell. Solutes above the membrane's molecular weight (MW) cut-off are retained in cell, while water and solutes below the cut-off pass into the filtrate and out of cell. The ultrafiltration discs (regenerated cellulose membranes YM-10 and YM-30 are frequently used) need a prewash/priming step to be used for the first time. After each use this type of membranes can be cleaned and reused until the membrane is free from any visible leak or scratch marks.
The detailed protocol for prewash, cleaning and storage are listed below.
1. Prewash: float membrane with glossy side down in beaker with distilled water for at least 1 hour changing water 3 times. All membranes are pretreated with glycerine to prevent drying, YM membranes are treated with sodium azide as a preservative.
2. Cleaning: rinse with 0.1M NaOH, followed by thorough flushing with distilled water. For strongly absorbed protein, soak in 0.1% protease solution and rinse thoroughly.
3. Storage: for reuse, store membranes in 10% ethanol water solution at 4°C.
Millipore (Amicon®) Ultra Centrifugal Filters/ micro-concentrators do not need any priming or prewash step for using for the first time. It comes ready to use and are not intended to be reused.
Amicon Stirred Ultrafiltration Cells are also used for protein concentration (RELATIVELY LARGER VOLUME), gas pressure is applied directly to ultrafiltration cell. Solutes above the membrane's molecular weight (MW) cut-off are retained in cell, while water and solutes below the cut-off pass into the filtrate and out of cell. The ultrafiltration discs (regenerated cellulose membranes YM-10 and YM-30 are frequently used) need a prewash/priming step to be used for the first time. After each use this type of membranes can be cleaned and reused until the membrane is free from any visible leak or scratch marks.
The detailed protocol for prewash, cleaning and storage are listed below.
1. Prewash: float membrane with glossy side down in beaker with distilled water for at least 1 hour changing water 3 times. All membranes are pretreated with glycerine to prevent drying, YM membranes are treated with sodium azide as a preservative.
2. Cleaning: rinse with 0.1M NaOH, followed by thorough flushing with distilled water. For strongly absorbed protein, soak in 0.1% protease solution and rinse thoroughly.
3. Storage: for reuse, store membranes in 10% ethanol water solution at 4°C.
The Amicon® Ultra concentrator samples are single use devices. There are 4 different sizes in this device family to accommodate various input volumes (0.5, 2, 4, and 15 mL). Another option for 70 mL is the Centricon® Plus-70 Centrifugal Filter Units. For these devices, a pre-wash is only used if glycerol interferes with downstream assays (glycerol used as wetting agent). Wash (using distilled water or buffer of choice) up to the device capacity volume and centrifuge until at least 50% has passed through membrane. Repeat wash if necessary to reduce glycerol levels more. Remove any remaining fluid from the device prior to loading sample. Note that if device is not being used right away, filter panels need to stay wet until use and then remove liquid rinse or storage buffer prior to use. A rule of thumb for selecting UF MWCO: Use a MWCO approx. 2x less than protein of interest size example use a 30 kDa cut off for a 66 kDa protein sample.
The Amicon® stirred cell is another option for larger volumes (>70 mL), and some membrane types can be used up to 5x. Note that the 0.1% protease solution may break down membrane components with long exposure times, but the general protocol Amritlal Mandal notes is fine.
If handled carefully, most of the associated membranes and devices can be reused, you just need to make sure that the membranes remain hydrated between use and that the same sample is used.
@Amritlal Mandal: For clarification, the EMD Millipore YM membranes do NOT contain sodium azide as purchased. Prerinsing is only necessary to remove glycerol humectant if this interferes with downstream applications.
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