FPLC Rules

Reserve System

Plan ahead

Sign up for your intended dates of use on the sign-up sheet located next to each FPLC.

The maximum length of sign-up time is 4 days during the week or 5 days including the weekend. You may sign-up again only on the last day of your currently scheduled run. Any exceptions must be cleared through the other lab users and/or Hans (e-mail is a good way to communicate).

Cancel any unused time if your plans change so another user may have access to the FPLC.

Sign the Column Log

Notebooks containing logs for each FPLC column are kept in Y4.212 (on the shelf over the FPLC parts cabinet).

Look up your column based on type and ID number and fill out the log sheet.

A column that is in long term use for a single protein may have one sheet filled out and labeled In Use.

To use the column labeled with someone´s name or initials you must first obtain their permission. You really should know how the column has been treated: what proteins, detergents, proteases, etc. may have been put on the column. You may realize you do not want the column after all.

Column Use

Please refer to the appropriate instruction sheet for your selected column. These instructions are also located on the shelf in Y4.212.

Basic rules:

• Never apply any cell lysates to the factory packed PFLC column (such as Mono beads or Gel Filtration). Therefore, at least one purification step must be done before using the FPLC column. Examples of first step columns are self-packed DEAE, SP Sepharose, Nickel, etc.
• Centrifuge or filter the sample before applying it to the column. This helps prevent frequent clogging of the column and excessive backpressure.
• Make certain that backpressure limits are correctly set for the particular column you are using. Set flow rates accordingly.
• It is best that columns do not leave the lab. Exceptions are when you work with collaborators. BUT the other lab members need to know who is removing the column from the lab, where the column will be used, and when the column is to be back in the lab and available for use.

Column Storage

• Do not leave a column dirty for the next person to use.
• If the column needs cleaning follow the appropriate method for that particular column. The instruction sheet for the column provides cleaning protocol.
• Refer to the appropriate instruction sheet for proper storage of the columns. Columns are usually stored in 20% ethanol, but some may be stored in 0.02% NaN3.
• Remove column from the FPLC and store at 4°C, if possible.

FPLC Cleanup

• Remove the column from the FPLC (did the column need to be cleaned first?).
• Remove the Superloop or sample loop from the system, if necessary. Store the Superloop dry or in 20% ethanol. Wash out the loading loop.
• Never leave buffer in the pumps unused for days. Pumps are stored in 20% ethanol.
• If you plan to clean a column do not fill the pumps with very concentrated acid, base or salt solutions.Use the injection port and loading loops to apply cleaning solutions to the columns.
• Leave the system tubing connected and filled with 20% ethanol. Never leave a flow cell dry because a dry flow cell can cause air bubbles later when it fills.
• Remove test tubes from the rack.
• Empty waste from the waste flasks.
• Remove buffer and accessories from the FPLC rack and work area.
• Clean up any drips or spills around the work area. Salts from the buffers cause corrosion.

No one wants to work in a sloppy sticky work area.

 

If you have any questions about how to use the FPLC or the policies or procedures please ask someone for assistance.