InstantBlue Protein Stain FAQs
What is the sensitivity of InstantBlue?
It can detect as low as 5 ng of protein (BSA).
Does InstantBlue has an upper limit of detection?
The stain does have an upper limit dependent on the volume of the InstantBlue Stain used. The more InstantBlue, the more Coomassie dye that is free to bind to protein. Once the Coomassie dye is exhausted, then it can’t stain anymore protein. If that is ever the case, just simply add more stain and more dye will be bound. Approximately, 20-25 mL of stain can stain up to 40 ug of total protein per lane (12 lanes per gel).
Is it possible to restain the gel to reveal what’s in the affected area? Also, would staining on a lab shaker eliminate the possibility of this happening?
Yes, they can restain with the same solution. A shaker would be a great and if staining for a long time (like overnight) we say to put a lid on the staining dish so nothing dries out or oxidizes.
It is written that no “fixing” of the protein is necessary with InstantBlue, the fixing occurs during the incubation with InstantBlue or whether no fixation at all is happening during the process?
With the InstantBlue the fixing is occurring during the incubation with the stain. The is the reason why in the manual and on the bottle we recommend staining for 1 hour. Although bands are usually visible after 15 minutes they are not fixed at this stage. This is fine for people who just want to take an image or cut bands out for mass spec for example. But if they were to transfer the gel to water after this time then the stain would diffuse away from the protein. After around 1 hour the stain will be fixed in the gel.
Why silver staining isn’t working following InstantBlue coomassie stain and wash?
Probably need to wash gel 3x 100 ml water before silver stain. They are likely staining SDS sequestering agent. They could also leave gel in stain overnight to see fainter bands, but still need water wash before silver stain”
Is there any test to check if the InstanBlue solution has been diluted?
Take a sample from a brand new unopened bottle and a sample from the suspect bottle. Then add BSA to both samples to a final concentration of 1mg/ml. Both samples should now be blue. Dilute this and measure the absorbance at 600nm in a UV spectrophotometer. You would need to experiment with the dilution ratio to get in the right range for the UV spec but it is going to be 10x or more. Both samples should be diluted the same amount. If UV absorbance of the suspect sample is much lower than the one from the new bottle then yes the suspected sample has been diluted.
Instant Blue was left out on the lab bench over the weekend. Please let me know if this will affect product performance or shelf life?
Being at room temperature for the weekend will not affect the product performance or shelf life.