Sample Preparation Guidelines for 2-Dimensional In Gel Electrophoresis (DIGE)

Sample preparation is a critical element in achieving optimal 2-D gel electrophoresis separation results. Hence, a discussion with an APAF staff member (from Array Technologies group) 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 frozen tissues or frozen liquid samples or dried down cell pellets.

APAF has a long experience in the preparation of samples from various sources for 2-D gel electrophoresis.For high quality sample preparations, essential for 2-D gel electrophoresis, we encourage to send original samples to APAF. However, sample preparation will incur a small fee. Sample preparation includes extraction of proteins, desalting, protein concentration, quantitation by Bradford assay and validation on 1-D gel. Please follow the general sample preparation guidelines for two-dimensional (2-D) gel electrophoresis. Some additional points to consider for DIGE gels are: extracted proteins (from either cells or tissues) OR lyphilised culture supernatant should be solubilised (at the final step) in 7M urea, 2M thiourea and 4% CHAPS sample solution. APAF will adjust pH of the samples just prior to labelling with CyDyes. Conductivity of the samples should be ~300 μS/cm. If sample has to be initially prepared (extract from plant or animal sources) other than the above-mentioned sample solubilising solution, it would be necessary to do a buffer exchange with the recommended sample solubilising solution. Ensure that the protein concentration remains within 5-10mg/ml.

There are some CRITICAL points for sample preparation for labelling with CyDyes:

• NO thiol reagents (e.g. DTT, TBP, beta Mercaptoethanol, etc.) should be present in any buffer prior to labelling.
• No primary amines (e.g. pharmalytes or biolytes, etc.) should be present in any buffer prior to labelling.
• Reduction and alkylation of proteins should be carried out only after labelling with the CyDyes.

The above-mentioned critical points are applicable for both Minimal and Saturation CyDyes.

Amount of proteins to supply

The amounts of proteins required for 2-D DIGE gels are as follows: 

Label Type	Protein quantity required
Minimal CyDye labelling	100 μg proteins per sample/per gel (17 or 18 cm long IPG strip)
Saturation CyDye labelling	5 μg protein per sample/per gel (17 or 18 cm long IPG strip)
Minimal CyDye labelling	150 μg proteins per sample/per gel (24 cm long IPG strip)
Saturation CyDye labelling	8 μg protein per sample/per gel (24 cm long IPG strip)

If you are unsure of protein concentrations in the protein sample (if proteins extracted by client) it is recommended to measure it with any available protein quantitation kit or by amino acid analysis (APAF is able to provide these services if client cover the cost). Correct measurement of protein concentration in a sample is very important for quantitative analysis. The following is a guideline to clients on how much sample to send (if APAF to prepare samples):

• Bacteria - approximately one-third of their dry-weight is proteins.
• Plasma/Serum - approximately 60 mg/ml protein (high abundance proteins correspond to 85 to 95% of the total plasma proteins).
• 1x10⁷Mammalian cells - approximately equal to 1 mg protein.
• Human tears - 1 μg of proteins in volumes between 0.2 μl -2.0 μl.
• Mammalian tissue (e.g. rat liver) requires a minimum of 10 mg freeze dried material.

• Human Urine between 10 ml to 20 ml needed for a large analytical gel ('normal' subject will require 20 ml per gel).

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 in our website or contact us in regards to permits needed for Australian quarantine regulations and the recommended couriers to use.

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.

2-D DIGE Gel Electrophoresis Service Request Form

Once you have followed all the guidelines you can download the 2-D DIGE Gel Electrophoresis Service Request Form from the website. Fill this form and send to APAF together with the samples.

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Last updated 10 Nov 2010