Frequently Asked Questions (FAQ's)

FAQ Topics

InstantBlue FAQ's

Q: Do I need to do anything prior to staining my gel? A: No, simply remove the gel from the cassette and place directly in the stain

Q: How long does InstantBlue take? A: You can expect to see the strongest bands appear after around 5 minutes with all bands being visible after 15 minutes. Total saturation of InstantBlue occurs after c.a. 1 hour.

Q: Do I still need to de-stain my gel? A: No, InstantBlue has been formulated not to bind to the SDS or Acrylamide in the gel, providing a clear background.

Q: How do I dispose of InstantBlue? A: InstantBlue is non-toxic and can typically be disposed of in the sink, however, always check this against your local regulations.

Q: Will my gels over stain? A: No, InstantBlue will not cause gels to be over stained, even if left submerged for days.

Q: What is the sensitivity of InstantBlue? A: InstantBlue can clearly visualise a 5 ng band of BSA, however, this may not be the case for your protein and depends on the basicity of your protein - the more basic the better the sensitivity. Refer to the table below to see where your protein fits with relation to BSA. Typically, staining is in the range of 5 to 25 ng protein per band

BradfordUltra FAQ's

Q: Can I use BradfordUltra if my sample contains detergents? A: Yes, BradfordUltra can resist the affects of detergents which normally influence standard Bradford assays.

Q: Which standards should I use for my standard curve? A: The Bradford assay is affected by the basicity and hydrophobicity of the protein. Typically, proteins have a basic amino acid composition of 10 to 17 mol% and you should try to use a standard as close to the mol% of basic amino acids as your unknown protein. Some good standards are shown below.

Protein Standard Mol % Positive Residues

Hen egg white lysozyme 13.9

Bovine serum albumin (BSA) 16.5

Bovine Immunoglobin (IgG) 11.3

Bovine beta lactoglobulin 11.8

NVoy Polymer FAQ's

Q: How tightly will NVoy bind to my protein A: NVoy associates very dynamically with proteins through hydrophobic interaction alone (as verified by null results using Isothermal Calorimetry). The exact strength of the interaction will be protein specific as shown by the Kd's calculated from the results of Surface Plasmon Resonance (Biacore) experiments for the interaction between NVoy and GFP (Kd = 28 uM) and NVoy and Hexokinase (Kd = 12 uM)

Q: How can I remove NVoy once I no longer need it in my sample? A: Typically, NVoy can be removed from your sample by binding it to an ion exchange or affinity column and washing with several column volumes of buffer not containing NVoy. Size exclusion / de-salting as a method for NVoy removal should be carefully considered as NVoy has a large hydrodynamic radius and may co-elute with your sample. HIC can be used as a polishing step during the removal of NVoy.

Q: Will NVoy affect my purification? A: NVoy should not affect either affinity or ion exchange purification. For size exclusion chromatography NVoy can be added to the sample to prevent Non Specific Binding (NSB) of the protein to the purification media, but it is not recommended to use it in the running buffer at concentrations above 1%. NVoy is not soluble in organic solvents and should not be use in Reversed Phase chromatography. NVoy will bind to Hydrophobic Interaction Chromatography (HIC) columns and this method can be used in the removal of NVoy from the sample.

Q: What amount of NVoy do I need? A: For an initial experiment to see if NVoy is going to be able to help we recommend a starting mass to mass ratio of 5:1 NVoy to Protein. This guarantees an excess of NVoy to protein so any effect should be measurable. Typically, the ratio can be optimised down to obtain the required effect.

Q: How long can I keep NVoy? A: NVoy is supplied lyophilised and in this state will last indefinitely. Once in solution, you should look to use within 1-7 days if stored at +4oC or 6 to 12 months if stored at –20oC or –80oC.

Q: How big is NVoy? A: NVoy has a molecular weight (MW) of 5 kDa. As it is a linear molecul it has a large hydrodynamic radius (RHYD) of ~18 kDa, which typically prevents NVoy from being able to enter the protein structure / binding sites unlike detergents and other additives.

RunBlue FAQ's