Uniblue A sodium salt

Uniblue A sodium is a reactive protein stain that can be used in the covalent pre-gel staining of the protein (Ex=594 nM)[1].

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Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)[1]. 1. Prepare the derivatization buffer: 100 mM NaHCO3, 10% SDS, pH 8-9. 2. Add 10 µL of 200 mM Uniblue A solution (dissolved in derivatization buffer) to 90 µL protein solution. 3. The samples are heated at 100°C for 1 min to perform the staining. 4. Add 100 µL of reducing solution composed of 10% glycerol and 20 mM dithiotreitol (DTT) in 200 mM Tris buffer with pH 6.8. 5. Excess Uniblue A reacts with Tris, resulting in a blue compound, serving as running front indicator in electrophoresis. 6. The sample is heated another minute at 100°C in order to achieve efficient reduction and allowed to cool to room temperature. 7. Add 20 µL alkylation solution containing 550 mM iodoacetamide (IAA), incubated for 5 minutes. 8. the samples are subjected to SDS-PAGE. Note: Dry protein samples or samples in compatible buffers (i.e. free of amines) can be diluted directly with the derivatization buffer solution to 5 mg/mL. In other cases, a prior trichloroacetic acid (TCA)/acetone precipitation or buffer exchange by ultrafiltration is recommended (see below). As positive control, bovine serum albumin (BSA) in derivatization buffer is used at a concentration of 10 mg/mL.

[1]. Marco A Mata-Gómez, et al. Accelerated identification of proteins by mass spectrometry by employing covalent pre-gel staining with Uniblue A. PLoS One. 2012;7(2):e31438.