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Q1 – Why is it better to use AcquaStain at room temperature?
We recommend allowing AcquaStain to equilibrate to room temperature before staining. AcquaStain works much more quickly at warmer temperatures. In fact it can take 10x longer to visualize bands using cold AcquStain. Read A2/Q2 for more detail.
Q2 – What is the optimal temperature for staining gels?
For achieving the most effective staining, we recommend preheating the appropriate amount of AcquaStain (25ml for a mini-gel) to a temperature between 37°C and 42°C.
Q3 – What does Acqua Stain bind to?
Acqua Stain binds to basic amino acids in a similar way standard coomassie does, only much faster and more sensitive.
Q4 – Can I further decrease the time for staining?
Yes. By placing the gel in Acqua Stain and microwaving for 10-20 seconds, you can cut the times by roughly 50%.
Q5 – Can I increase the sensitivity/contrast on my Acqua Stain Gels?
Yes. If you gently agitate the gel in stain at 50°C during staining, you will see increased sensitivity. If you gently agitate the gel in heated deionized water (50-60°C) for 15-25 minutes post staining, you will see increased contrast.
Q6 – Why do the protein bands on my gel appear as blobs when I incubate overnight?
It is not possible to overstain however, Acqua Stain is more sensitive than traditional methods so it is quite possible that the gel was overloaded. In addition, low molecular weight proteins can diffuse over time if the gel isn’t fixed. We recommend decreasing the stain time to 60 minutes or less or loading less onto the gel.
Q7 – Does Acqua Stain fix the gel?
No. Acqua Stain is an aqueous-based stain and therefore will not fix your gel. After 30 minutes of incubation with the stain, the gels are very stable so there is no need to fix the proteins.
Q8 – After staining, can I store the gel in acetic acid?
Yes. You can store the gel in acetic acid or deionized water.
Q9 – Can Acqua Stain be used in colorimetric assays?
Yes. You can measure with a standard spectrophotometer or plate reader at 595nm
Q10 – What is the protocol for using proteins stained with Acqua Stain in mass spec analysis?
1) Cut out protein band and place in microfuge tube
2) Add 1 ml 30% ethanol or 30% acetone (30% acetic acid may be used but will result in acetylation of N-terminus).
3) Incubate for 30 minutes.
4) Repeat steps 2 and 3 until all stain removed from protein (typically 2 or 3 washes are required for complete stain removal)
5) Process as typical for mass spec analysis
Q11 – What is the protocol for drying down the gels after staining?
1) Ensure that the gel has been stained with Acqua Stain for at least 1 hour.
Although protein bands will be visible after a few minutes of incubation in stain,
the staining process is completely stable after 1h of incubation. Depending
on the type of gel you are using longer incubation may be necessary. Further
processing of the gel prior to completion of the staining process may result in
protein destaining and reduced sensitivity. If this occurs simply restain the gel
by incubating overnight in Acquastain
2) Submerse the gel in approximately 100 ml ultrapure water at ~70°C (heat for
30s to 60s in a microwave oven). Incubate for at least 1 hour while gently
rocking. Optionally adsorbent paper or paper towel can be added. Gels can be
incubated overnight in water.
3) Incubate the gel in a ‘gel drying solution’ (e.g. 4% glycerol, 20% ethanol in
water) for 2 minutes. Incubation of any Coomassie-stained gel in an alcohol
solution will eventually result in destaining of the bands so avoid incubation for
longer than 5 minutes.
4) The gel is now ready for drying between wetted cellophane membranes.
Q12 – Is AcquaStain compatible with a western blot?
Yes. Because it reversibly binds, you can use it for a western blot. You will need to wash the gel a few times in transfer buffer. You can also stain the gel after blotting as this is a good method of determining transfer efficiency.
Q13 – Can AcquaStain be reused?
Yes. Typically, customers can use it two or three times.
Q14 – Can AcquaStain be followed by traditional silver staining?
Yes. Please wash 3x with 50% ethanol for 15 minutes before proceeding with a standard silver staining protocol. Expected results shown: