2-Dimensional Gel Electrophoresis Sample Preparation Guidelines

Sample preparation is a critical element in achieving optimal 2D electrophoresis separation results. Hence, discussions with an APAF staff member concerning the nature of samples to be sent for analysis is highly recommended.

Sample Preparation

Where possible it is advisable that samples are sent to APAF in their original form. For example as frozen tissue or dried down pellets. Where this is not possible samples should be supplied free of the following components which interfere with the first dimension isoelectric focusing step:

•   salt
•   nucleic acids
•   polysaccharides
•   lipids

 

Salt is a particular problem which leads to burning of IPG strips and we recommend the following desalting methods:

• Organic solvent precipitation
• TCA/acetone precipitation
• Buffer exchange using 5 kDa cutoff membrane 
• Dialysis

Note: If additional work needs to be done at APAF to reduce conductivity (acceptable conductivity is <300µS/cm) of the supplied sample prior to the first dimension separation then additional costs will be incurred.

Note: If proteins are extracted from any cell or tissue it is VERY IMPORTANT to add protease inhibitor cocktails during lysis.

Note: If sonication/homogenization is required to extract proteins it important to keep samples on ice in between steps to avoid any potential degradation and carbamylation of proteins.

Note: APAF offers services for High Abundance protein removal from both human and rodent plasma samples using antibody affinity chromatography based technologies. Clients are requested to contact APAF for any specific information required for this service and for the cost associated with it.

 

Amounts of protein to supply

 The following table provides a guideline to the amounts of protein required (per gel) for various 2-D gel types:

Analytical load (fluorescent stain) mini gel	10-50 μg total protein
Analytical load (fluorescent stain) large gel	100-300 μg total protein
Preparative load (coomassie) mini gel	100-200 μg total protein
Preparative load (coomassie)	1-2 mg total protein

 

 Note: protein load varies depending on the pH range of IPG strip.

 If you are unsure of protein concentrations and need to know how much sample to send, the following is an approximate guide:

• Bacteria are approximately one third their dry-weight in protein
• Plasma/Serum is approximately 60mg/mL protein
• 1x10⁷ Mammalian cells is approximately equal to 1mg protein
• Human tears contain 1 μg of protein in volumes between 0.2 μl-2.0 μL
• Mammalian tissue (eg rat liver) requires a minimum of 10mg freeze dried material
• Human Urine anywhere between 10mL to 20mL needed for a large analytical gel ("normal" controls will require 20mL).

Samples prepared for 2-D gel can be used for 1-D gel but not vice versa.

Pathogenicity  

Biological samples coming into APAF should be accompanied by documentation of potential pathogenicity or pathogen free status otherwise APAF will presume all samples from human and animal origin are potential pathogens and will be treated accordingly.

How do I send samples from Overseas to APAF?

If you are sending any biological samples to APAF from countries outside of Australia go to the Quarantine guidelines on our website or contact us in regards to permits needed for Australian quarantine regulations and the recommended couriers to use.

Sample Request Forms

Once you have followed all the guidelines you can download the 2D Gel Electrophoresis Request Form.

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Last updated 05 Nov 2010