1.3.1. Preparing samples
Ideally, samples should contain about 1 - 10 µg of total protein to give optimal results, thus protein content of the samples should be assessed by one of the methods described above (Bradford or BCA, section 1.2). Haemolymph proteins are usually best separated in gels with a 7.5 % acrylamide concentration.
1. Prepare the sample buffer by dissolving 1.51 g Tris, 20 ml glycerol in 35 ml of double distilled water (ddH2O).
2. Adjust pH to 6.75 with 1 N HCl.
3. Then add:
3.1. 4 g SDS,
3.2. 10 ml 2-mercaptoethanol,
3.3 0.002 g bromophenol blue,
3.4. ddH2O to a final volume of 100 ml.
4. Prepare protein samples (haemolymph) by adding the sample to the sample buffer in a 1:1 (v/v) ratio.
Very diluted samples may require a 2X concentrated sample buffer to make an adequate volume of 10 – 15 ml.
5. Prepare a sample containing the molecular mass markers, following the manufacturer’s instructions.
Use the same sample buffer (step 1) as that used for your samples.
6. Heat samples and the molecular mass marker sample in a boiling water bath for 1 – 3 min to denature protein structure.
Perforate tops of Eppendorf tubes to avoid popping of the lid and spilling of the sample as internal pressure increases with heating.
7. Cool on ice for a few minutes.
8. Spin in a refrigerated tabletop centrifuge at maximum speed for 5 min.
9. Use supernatant only for application to the gel, as the precipitate may contain protein and nucleic acid aggregates which may cause streaking along the separation path.
User safety: Mercaptoethanol is an irritant and a foul smelling compound. It can be substituted with dithiotreitol.