Guide on our SEC-MALS

SEC-MALS is short for size-exclusion chromatography-multi-angle-light scattering.

Our setup is designed for analytical scale analysis of your purified proteins to obtain information on properties like oligomeric state and aggregation.

Our setup is composed of a Dionex HPLC that consists of a pump, autosampler, multiwavelength DAD that is coupled to a 3-angle MALS detector from Wyatt. This setup allows for the separation on-column by size exclusion followed by detection via UV and MALS.

More background on MALS can be found here in the attached documents.

General MALS theory (Wyatt)

SEC-MALS (Wyatt)

Overview of a SEC-MALS run (11.11.21 KL)

Start-up

From a off state:

Turn on:

• Pump module
• Autosampler module
• Column module
• DAD module
• Wyatt MALS

When you come lines A, B and C should always be in 20% Ethanol. Line D should be in water or in 20% Ethanol. The extra bottle is the pump wash. This should also be in 20% ethanol

MOST IMPORTANT THING

ALL BUFFERS NEED TO BE FILTERED 0.1 um. First filter 0.45 um then 0.1 um. Buffers last maximum 2 days, and then need to be refiltered. Unfiltered buffers will clog the detectors.

Recommended buffers are 1X PBS or 150 mM NaCl, 10 mM HEPES 7.5. Any other buffers please ask first.

Water can be taken directly from the MQ machine. This has already been filtered through the pod.

Turn on the Chromeleon program. This is the program that controls the HPLC.

Pump purge

• Open casing of the pump module. Loosen the golden knob
• On the pump tab of Chromeleon, change to 100% D if its not already there.
• On the left, click Pump purge. This will take 5 minutes.
• When the purge is complete, tighten back the golden knob.

Equilibrate the column

• We are using a standard column:XXXXX It has a flow rate of X mL/min. A column length of X mL. and a max pressure of X mPa
• With pump D at 100%, set the flow at X mL/min. The pump panel on the pump and on the pump tab on Chromeleon should show the current flow. If there is no flow, the HPLC may be "stopped" red circle. Click on the green triangle to make it "Go"
• Equilibration should be performed until UV baseline is flat and the MALS detector has noise in the fifth decimal place ie. 0.0000X

Turning on UV detector and MALS detector

• On the DAD panel in Chromeleon turn on the UV lamp (no need for Visible lamp unless you are looking at wavelengths +650 nm). It takes a few moments before it is lit.
• When it is lit, click on 'Monitor baseline' to start looking at the baseline
• Next click on the XXX settings, make sure XXXX

• Turn on the MALS detector by click on the LAMP button on the screen to turn it back on. The system will put it Idle/Off within 30 minutes.

Starting a run

• Once the column is equilibrated (low noise and flat baseline) You are ready to experimentg.