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Frequently Asked Questions

General Assay :

Is the room temperature important when incubating the plate in the final step before reading the fluorescence?
A: Yes, room temperature is important and should be about 25 °C. If the room temperature is below 20 °C , the assay performance will be affected.

What is the rotator mentioned in the protocol?
A: The rotator refers to a machine that rotates with different angles to allow media to move in each well. A shaker should also work as long as there is gentle shaking.

If the assay doesn’t work for me, do you offer technical support? 
A: Yes, if you have any questions, you may ask us anytime at [email protected]. Out team is always glad to help !

 

Plate Reader and Microplate Settings:

What are the required settings for the plate reader to use the Bridge-It® Assay?
A: First, you would need a plate reader that is equipped for FRET (Fluorescence Resonance Energy Transfer). Then, you would need excitation filter at ~480-485 nm and emission filter at ~520 – 540 nm (for cAMP and cAMP-PDE Assays) and 665 nm (for other assays including SAM, L-Trp and L-Met). The plate should be read from top and plate reading settings of 50 times per well is recommended.

What are the required settings for the plate reader to use the FRET- PINCER® Assay?
A: First, you would need a plate reader that is equipped for FRET (Fluorescence Resonance Energy Transfer). Then, you would need excitation filter at ~485 nm and emission filter at ~540 nm (for donor signal) and 665 nm (for acceptor signal). The plates should be read from the top and plate reading settings of 50 times per well is recommended.

What are the required settings for the plate reader to use the TR-FRET Assays (TR-FRET Bridge-It® and TRF-PINCER®)?
A: First, you would need a plate reader that is equipped for TR- FRET (Time-Resolved Fluorescence Energy Transfer). Then, you would need excitation filter at ~330 nm and emission filter at ~620 nm (for donor signal) and ~665 nm (for acceptor signal). The plate should be read from top. Read settings at 50 times per well and delay time of 55 μs are recommended.

Do you have a suggested band pass for the excitation and emission filters?
A: Excitation and emission filters with varying bandpass are available and differs from vendor to vendor. However, we have been using the filter with following settings:

For TR-FRET based assays: (TR-FRET Bridge-It® and TRF-PINCER®):

330 nm excitation filter (band pass: 80 nm)
665 nm emission filter (band pass: 7.5 nm)
620 nm emission filter (band pass: 40 nm)

For FRET based assays: ( Bridge-It® and FRET-PINCER ®):

485 nm excitation filter (band pass: 20 nm)
665 nm emission filter (band pass: 34 nm)
540 nm emission filter (band pass: 40 nm)

 

What plate gain settings or sensitivity should be used?
A: Gain settings and sensitivity settings vary from one plate reader to other. Gain setting between 2,000-60,000 is fine.

Does the type of microplate used to read the fluorescence matter?
A: Yes, you must use black non-binding surface plates to perform the assay, and read fluorescence from the top. A black non-binding surface microplate is recommended for following reasons:

  • Black walls reduce well-to-well crosstalk and autofluorescence during fluorescent assays

  • Non-binding surface creates a nonionic hydrophilic surface that minimizes molecular interactions with the plate surface

One plate is already provided in the assay kit. If you need more microplates, you may buy either the 96-well (cat#: 163300) or 384-well (cat #: 163301) from us.

Assay Specific Questions:

S-Adenosyl Methionine (SAM) Assay Kit:

Is it possible to use cell extracts for quantifying SAM levels? How to perform extraction from cells?
A: Yes, we have tested the cell extracts using human cells (HEK293 and HeLa). Please contact us for details .

Should I increase the amount of extraction buffer (Buffer CM) depending on the number of cells?
A: Extraction buffer can be 30 ul for 25,000-50,000 cells. You may use 150-400ul for 250,000 cells.

Does the SAM Assay Solution have to be incubated at 37°C for 30 minutes?
A: Incubating the assay solution at 37°C for 30 minutes gives more reproducible results, however you may thaw at room temperature (25°C) if preferred.

How do I determine the concentration of SAM in my samples?
There are many factors like growth conditions, type of cells and age of cells that might affect the SAM level in cells, so there is not a definite way to estimate the SAM level beforehand. A method to quickly get a rough estimate of SAM level would be to make a series of dilutions of your sample (For eg: 1:10, 1:20 ,1:40, 1:50 ,1:100 etc.) and see which of the readings fall within the range of the Assay standard curve using the Bridge-It® Assay. Then, to get an accurate estimation of SAM in your sample, you could work with whichever dilution falls in the range of the Assay standard curve in duplicates or triplicates.

L-Tryptophan Fluorescence Assay Kit:

I ran out of Buffer W. Can I use something to replace it?
A: Yes, you can use the following buffer to dilute your sample: 20 mM Tris (pH = 8.0) containing 1 mM EDTA.

Can I quantify Tryptophan in human plasma samples using this kit?
A: Yes, ours kits has been successfully used to measure Tryptophan in human plasma samples with high  recovery and reproducibility .Please contact us for details.

Can I use your Bridge-It® Tryptophan Assay Kit to measure total tryptophan?
A: Yes, we have tested our Bridge-It® kits to measure total tryptophan on human plasma samples and human cell pellets with excellent recovery and reproducibility (HEK 293).

Can I refreeze the kit reagents after I use them?
A: Buffer W and the Tryptophan Standard can go through 5 freeze/thaw cycles. Assay Solution A can go through one freeze/thaw cycle and then can be reused for up to a week stored at 4°C.

cyclic AMP (cAMP) Assay Kit:

Can I refreeze the 10X KRB-IBMX in aliquots after I use it the first time?
A: Yes, you can freeze and thaw the 10X KRB-IBMX buffer many times without affecting the activity of solution. If you see precipitates after thawing, warm up to 37 °C to dissolve.