Gel Band Proteins:
For protein antigens that are difficult to purify, running the protein on a prep gel is a quick and popular approach for isolating the protein of interest. Even better, the target band can be cut out and used directly for immunizations. This is a popular and effective approach, but please keep the following guidelines in mind:
- For the best results, protein concentration within the gel band should be as high as possible to ensure that optimal quantities of protein are being immunized. We recommend aiming for 250 µg/ml as the lower limit – anything higher will be safe and is welcome.
- Gel bands should be stained, de-stained, rinsed well and trimmed to avoid as much extraneous material as possible.
- Please do not chop, mash or homogenize the gel bands.
- Please do not elute the protein from the gel bands – this will introduce SDS into the mixture and prevent the formation of an emulsion with the adjuvant. The SDS present in the intact gel bands does not present this problem.
- The protein quantity / concentration can typically be determined by measuring the total protein quantity prior to running the gel and then estimating the concentration of protein in the target band based on its relative size compared to any other bands. For example if 5 mg of total protein is loaded on a preparative gel and it’s estimated that the target band accounts for approximately 60% of the total protein bands, then 3 mg of the target protein should be present in the cut-out bands. An exact concentration isn’t necessary, only a good estimate.
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