Biotechnology
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Frequently Asked IRDye® Questions
1) What NHS ester dyes are recommended for the 700 channel of LI COR instruments?
2) What NHS ester dyes are recommended for the 800 channel of LI COR instruments?
3) What dye should I use in place of IRDye® 38 / NN382?
4) What dye should I use in place of IRDye® 78?
5) What dye should I use in place of IRDye® 80 / IRDye® 75?
6) What dye should I use in place of IRDye® 40 / IRDye® 41?
7) Can I use these dyes to label unmodified DNA/RNA?
8) Is "NN382" available?
9) I really need (discontinued dye). Is there any way I can get some old samples?
10) Are any other reactive groups besides NHS ester available?
11) Are any of the dyes suitable for Fluorescence Polarization (FP) experiments?
12) What are the fluorescence lifetimes of the dyes?
13) What are the "correction factors" for 260/280 nm for the dyes?
14) Can I do the labeling reaction in organic solvents (or organic aqueous mixtures)?
15) I did the labeling in DMF and got a low yield of labeled product. What's wrong?
16) How long can the dye NHS esters be kept at -20C (or -80C)?
17) What is meant by "dye stacking?"
18) What DTT concentration can be tolerated?
19) What QC tests are done for the dyes to make sure they are "good?" Is there a functional test to prove the dyes can label things?
20) My HPLC shows two components in the dye. Why is that?
21) Are there antibodies raised to our IRDye® Infrared dyes?
22) Can a virus be labeled for single shot (replication defective) virus gene delivery vectors?
23) What dye should be used to label carbohydrates?
24) What IRDye® 800 dye should be used to label DNA?
25) What IRDye® should be used to label DNA?
26) What dye should be used to label peptides?
27) What dye should be used to label proteins?
28) What IRDye® 800 dye should be used to label RNA?
29) What dye should be used to label RNA?
30) Does a higher dye to protein ratio increase the fluorescence?
What NHS ester dyes are recommended for the 700 channel of LI-COR instruments?
From LI-COR: IRDye® 700DX
From LI-COR: IRDye® 680 [top]
What NHS ester dyes are recommended for the 800 channel of LI-COR instruments?
From LI-COR: IRDye® 800CW and IRDye® 800RS.
In general a dye can be used, provided it can be excited at 780 nm and detected at 820 nm. [top]
What dye should I use in place of IRDye® 38 / NN382?
The IRDye® 800CW NHS ester is recommended.
Conjugation conditions will have to be adjusted, due to differences between NHS ester chemistry and isothiocyanate (ITC or NCS) chemistry of the old dyes. Follow the instructions for the new dye. [top]
What dye should I use in place of IRDye® 78?
The IRDye® 800CW NHS ester is recommended. This should "drop in" to previous protocols you have been using. [top]
What dye should I use in place of IRDye® 80 / IRDye® 75?
The IRDye® 800RS NHS ester is recommended. This should "drop in" to previous protocols you have been using. [top]
What dye should I use in place of IRDye® 40 / IRDye® 41?
There is no direct replacement. The IRDye® 800RS NHS ester is the closest. Conjugation conditions will have to be adjusted, due to differences between NHS ester chemistry and isothiocyanate (ITC or NCS) chemistry of the old dyes. Follow the instructions for the new dye. [top]
Can I use these dyes to label unmodified DNA/RNA?
No. The NHS ester group will not react with ordinary nucleic acids. [top]
Is "NN382" available?
No. It is the same dye as IRDye® 38, now discontinued. [top]
I really need (discontinued dye). Is there any way I can get some old samples?
No, discontinued dyes are no longer available. [top]
Are any other reactive groups besides NHS ester available?
Contact us using the form on this web site. [top]
Are any of the dyes suitable for Fluorescence Polarization (FP) experiments?
Yes. IRDye® 700DX is suitable for FP. [top]
What are the fluorescence lifetimes of the dyes?
For the two 800 dyes, approximately 800 psec. For IRDye® 700DX, approximately 1.5 nsec or longer. [top]
What are the "correction factors" for 260/280 nm for the dyes?
For each dye below subtract this percentage of the absorbance at the dye maximum:
IRDye® 800CW = 3.0%
IRDye® 800RS = 3.5%
IRDye® 700DX = 9.5%
There is no significant effect of solvent or difference between 260 and 280 nm. [top]
Can I do the labeling reaction in organic solvents (or organic aqueous mixtures)?
Yes. DMSO is preferred. Do not use DMF. There are traces of dimethyl amine in DMF, even the purest grades. [top]
I did the labeling in DMF and got a low yield of labeled product. What's wrong?
The dimethyl amine produced by DMF decomposition reacts with NHS esters. When the dye concentration is low (ca. 1 mg/ml or less) traces of amines can react with all the NHS esters. [top]
How long can the dye NHS esters be kept at -20C (or -80C)?
The unopened dyes in the original containers, are good for one year. Solutions of the dyes, even frozen, may lose reactivity much faster. [top]
What is meant by "dye stacking?"
Most dyes have some tendency to form dimeric structures in solution.
See: W. West and S. Pearce, J. Phys. Chem., 69, pp 1894-1903 (1965) [top]
What DTT concentration can be tolerated?
There is no general answer to this. It will depend on the concentrations of dye and DTT, time, and temperature. Sequencing with IRDyes™ works, despite the presence of DTT in the enzyme buffers. [top]
What QC tests are done for the dyes to make sure they are "good?" Is there a functional test to prove the dyes can label things?
The bulk dye is analyzed by HPLC, UV/vis, and NMR spectroscopy to confirm identity and purity. The packaged dye is analyzed by HPLC and UV/vis to confirm the amount of intact NHS meets specifications. No functional test is needed [top]
My HPLC shows two components in the dye. Why is that?
There is always some of the carboxylic acid dye present. It elutes before the NHS ester in reverse phase HPLC. The specification and the amount printed on the package are based on the amount of active NHS ester present. [top]
Are there antibodies raised to our IRDyeŽ Infrared dyes?
No. [top]
Can a virus be labeled for single shot (replication defective) virus gene delivery vectors?
Nucleic acids can only be labeled if they have been modified/synthesized with an amino group to react with the NHS esters. [top]
What dye should be used to label carbohydrates?
If the carbohydrate has been modified to contain an amino linker, IRDyeŽ NHS Esters should work. [top]
What IRDyeŽ 800 dye should be used to label DNA?
This requires functionalization of the DNA with one or more amino-linker groups. IRDyeŽ 800RS is usually the best choice. This will give the easiest purification of the product (by HPLC). If there will be many dyes per DNA strand, IRDyeŽ 800CW will minimize the interactions between dyes, which could improve performance. [top]
What IRDyeŽ should be used to label DNA?
If the DNA has been modified to contain one or more amino linkers, IRDyeŽ NHS Esters should work. [top]
What dye should be used to label peptides?
For the 800 channel, use IRDyeŽ 800CW. For the 700 channel, use IRDye 700DX. [top]
What dye should be used to label proteins?
For the 800 channel, use IRDyeŽ 800CW. For the 700 channel, use IRDye 700DX. [top]
What IRDyeŽ 800 dye should be used to label RNA?
This requires functionalization of the RNA with one or more amino-linker groups. IRDyeŽ 800RS is usually the best choice. This will give the easiest purification of the product (by HPLC). If there will be many dyes per RNA strand, IRDyeŽ 800CW will minimize the interactions between dyes, which could improve performance. [top]
What dye should be used to label RNA?
If the RNA has been modified to contain an amino linker, IRDyeŽ NHS Esters should work. [top]
Does a higher dye to protein ratio increase the fluorescence?
Yes, in general, if the D/P is not too high. However, as the dye groups on the probe get closer together, they interact with each other, reducing the fluorescence. Thus crowding the dyes together by over labeling can give a less fluorescent product. Too many labels can also reduce a probe's activity and/or cause it to bind non-specifically. [top]
