Notes for Successful Chromatography

Sample Preparation for chromatography

Most commonly used technique to clarify the lysate is to centrifuge it at high speed.Protein solution can be filtered to remove particulates from the solution which otherwise block the flow in the column.Filters used should be low protein binding membranes like the PVDF and PES.Loss of protein yield can occur if nylon filters are used. 0.45 µm or 0.2 µm filters are generally used for filtering if there is restriction in the flow through the filters 0.8 µm filters can be used.

While preparing sample for ion exchange chromatography make sure the conductivity is below 10 mS/cm, which will ensure optimal binding.

Proper protein storage methods after purification can increase the stability of proteins.

• Short term (24 hr or less): 4°C
• Long term: -80°C. Include 5–50% glycerol, albumin (10 mg/ml), and/or reducing agents like DTT or BME in the storage buffer. Freeze protein in small aliquots in microcentrifuge tubes.
• Flash freeze tubes using liquid nitrogen, or dry ice/ethanol bath
• Proetin can also be stored as ammonium sulfate precipitate at 4°C

Column / System Check

Operating pressure is important factor which can influence the chromatographic resolution.Make sure the column is run at the correct pressure. Operating pressure will be mention in the chromatography media manual.

Height equivalent theoretical plate (HETP) count and asymmetry factor (As) are dependable measures of column performance. Note these parameters before first column use to enable

monitoring over time. Always make sure to take your column out of line or put your valves in column bypass prior to purging.

Replace worn frits and O-rings in manually packed columns when they are observed.When attaching a column to your system, you should use the drop-to-drop method to avoid introducing air onto your column. This is a method of filling the column inlet with fluid prior to final attachment.

Occasionally check your UV detector lamp life to ensure it meets recommended output parameters. Tubing on your chromatography system should be short enough to minimize dead volume while allowing component positioning flexibility.Buffer lines should be cleaned with purified water at the end of the day. This is especially important when working at low temperatures where salts can precipitate.

Degas or vacuum filter buffers prior to use to minimize air and particulates in the mobile phase.Occasionally inspect buffer bottles/bags for signs of bacterial growth. Solutions that appear cloudy upon shaking or stirring should be discarded. Treatment with a bacteriostatic agent such as 0.02% sodium azide will prolong solution storage.

Sample inlet lines and sample loops should periodically be cleaned with 1 N NaOH solution, followed by water or neutralizing buffer, to eliminate sample buildup in the tubing. Periodically replace PEEK tubing that comes in regular contact with sample, especially lines connected toyour fraction collector.

Storing all system lines in fresh 20% ethanol when not in use is an effective way to avoid bacterial growth. It is recommended that this is done at the end of each work day.Injection needles are best used with volumes of 1 ml or less. Larger volumes can be more easily managed with a luer fitting attached to your injection port. Calibrate your pH probe daily for most accurate results. Electrodes should always be stored in their designated storage solution or a neutral buffer. 20% EtOH should not be used, as it changes the concentration of KCl in the probe.

Pre-equilibrate the selected column prior to running and observe UV/conductivity behavior stability. Many automated systems allow you to advance to sample injection once complete equilibration is observed.Periodically check system tubing for cracks and bends.

Tubing bends can restrict flow and increase system backpressure.For best results, occasionally sanitize your entire system with 1 N NaOH to clear the flow path from pump to fraction collector.

Detectors

Random spikes in a UV trace that coincide with negative conductivity spikes most often indicate presence of air. Recurring instances of these spikes can often be corrected by purging your system with 1 N NaOH.Signs of pulsation in your gradient may indicate that you are not using the correct size of mixer for your flow rate.S-shaped conductivity gradient curves often indicate a mismatch between mixer size and flow rate.Sawtooth UV signals can be an indication of air trapped in your flow cell. You can often chase the bubble out of your system with 1 N NaOH.

Loading lysate through sample pumps is preferable over using system pumps. The latter increases the chances of sample buildup and contamination in your system.To achieve optimal gradient performance, choose the mixer size that corresponds with the operating flow rate. Proteins and nucleic acids can be monitored together with a multi-wavelength detector. You can also analyze their relative proportions using software for automated chromatography systems.There's a

Know your Protein

Know relevant properties of your protein: pI, molecular weight, hydrophobicity. This will assist in developing a purification strategy. If using IMAC as a capture step, do not add EDTA to your buffers. These will strip the active metals and preclude protein binding.

When creating methods try to minimize redundant steps for clarity of protocol and ease of monitoring. Creating method templates saves future setup time and eliminates the need to monitor entire runs.Tris-based buffers should not be used in IMAC purification, as they will compete the protein of interest for the binding sites.Do not use phosphate buffer systems when usinghydroxyapatite resins, as they will interfere with protein binding. Always name your runs by date or notebook number to facilitate easy data access in the future.

References

Chromatography Success tips and tricks guide for successful preparative chromatography and Protein purification, Biorad

Protein Purification Handbook, GE

Stratagies of Protein Purification,GE