Tetramer Preparation: MonoQ Purification of Biotinylated MHC/peptide Monomers

Preparation

Check the levels of Buffer A (20mM Tris, pH 8) and buffer B (20mM Tris, pH 8, and 500mM NaCl).
Attach the 2ml super loop to valve 1.
Note: When working at the bench, keep the sample and all buffers on ice (~4°C).

Turn keypad on.
Press [manual] button on the FPLC to turn the keypad on.
Close valve 1.
Press [step forward] button three times to open the valve mode. Close valve 1 by setting it to position 2 by entering [1.2] followed by [store].
Remove the needle from the injection port.
The needle can only be removed from the injection port when valve 1 is closed (i.e. in position 2). Closing valve 1 prevents air from entering the line and subsequently being injected into the column.
Wash, fill and insert the needle into the injection port.
Wash a 3ml syringe with 20mM Tris (pH 8) thoroughly. Fill the syringe with 2ml of 20mM Tris (pH 8), remove all excess air bubbles and insert the needle into the injection port.
Open valve 1 and inject the sample.
Open valve 1 by setting it to position 1 (press [1.1] then [store]). Inject the 20mM Tris into the 2ml loop being careful not to bend or break the needle.
Set the flow mode.
Press the [step backward] button three times to the flow mode (ml/min) and set the flow by pressing [4.0] then [store].
Close valve 1.
Press the [step forward] button to the valve mode and close valve 1 by setting it to position 2 (see above instructions).

The loop should drip at a rate of 4ml/min.
Repeat wash.
After the loop has been washed, remove the syringe and fill it with 2ml of 20mM Tris (pH 8.0). Repeat the above procedure for washing the loop.

Close valve 1, fill the needle and insert it into the injection port.
Press [step forward] button three times to open the valve mode. Close valve 1 entering [1.2] followed by [store].

Remove the syringe from the injection port and fill it with 500ml 20mM Tris (pH 8.0) and the centrifuged biotinylated sample from the buffer exchange (concentrated to 500ml). The total volume to be injected is 1ml.
Open valve 1 and inject the sample.
Insert the syringe into the injection port and set valve 1 to position 1. Inject the sample into the super loop and press [end].
Set up fraction collector rack.
Fill the large fraction collector rack with 42 disposable 1.5ml polypropylene tubes. Place the rack on the fraction collector. Align the arm such that the middle of the arrow is against the center of the first tube.
Set the fraction collector.
Set the fraction size by pressing [fraction size], [1] and [store return].
Set the peak fraction size by pressing [peak frac. size], [1] and [store return].
Set the delay by pressing[delay], [0.34] and [store return].
Run MonoQ program.
Open the FPLC computer program to run the MonoQ column. From the main menu, the program is located in the “MonoQ” folder and entitled “MamuA11.”