[help] about plate reader wavelength (View forum version)



N_L

Posted 19 January 2011 - 09:02 PM

Hi.
I was planning to run Glutathione assay kit for my project using following kit.
http://www.caymanche...alog/703002/a/z

Unfortunately, I found out that 405nm filter on my plate reader is faulty (Lamp energy too low) and cannot be used.
But I cannot wait to get it repaired. Their recommended range is 405-414nm.
Which other wavelength can I try instead of 405? I have options of 340, 450, 490, 540, 595, 750nm to choose from.
What possible things can happen if I use other filter other than recommended nm? But I'm willing to take the risk.

Any advice is appreciated.

protolder

Posted 19 January 2011 - 10:34 PM

Hola, If you have the enzyme , substrate and DTNB, you could made the reaction in a tube, stop it when the colour were appreciable and made an absortion spectrun in any spectophotometer to see the maximun of absorbance, wich will be at 405-410nm, comparing the response at the others wavelengst, you could choice the second abs were the response were appreciable . If you only have coated plates, use a column of it to made a control reaction with increasing amounts of sample or with the same amount and increasing times, and read it at the different absorbances looking for that in wich the relations betwen wells is corresponding to the variable studied. Buena suerte

sgt4boston

Posted 20 January 2011 - 03:32 AM

just run a few point of your curve low mid and high at 340 and 450 select the wavelength that gives you the best response. I think 450 would be best as it is the closest.

bob1

Posted 20 January 2011 - 04:13 PM

It all depends on the absorbance curve for the assay as to whether using other wavelengths is appropriate as not all parts of the mixture will absorb at the same ratios of substrate:product. You could try ringing/emailing the company to see if there are other wavelenghts that can be used.

DRT

Posted 23 January 2011 - 02:06 PM

If it is the ‘lamp energy’ then you may need a new light source not a new filter.
But in addition to the recommendations above; you ought to be able to continue with the 405nm so long as you have an appropriate standard curve albeit with reduced readings and sensitivity.

good luck

N_L

Posted 26 January 2011 - 07:29 PM

Thank you, all.
I also contacted technical support of the kit and replied to try at 450nm(closet to 414), though with reduced sensitivity. Anyway, it worked more or less.

@DRT, Sorry, I don't get about difference between light source vs filter.
In that machine, only 405nm doesn't work. Other filters (450, 595, 750) just work fine.
Anyway, I contact the instrument vendor and they said; need to change the lamp.

bob1

Posted 27 January 2011 - 03:17 PM

The lamp energy is a measure of the amount or "strength" of the light coming out of the light bulb in the machine. What you perceive as white light is actually made up of lots of different wavelengths, some stronger and some weaker in output (see: Sodium bulb emission spectrum. As the bulb gets older, the bulb produces less light, some of which happens to be the at the 405 nm wavelength. The machine calibrates the amount of light it receives from the bulb and uses this as a basis for measurement... if too little light is received, it can't calibrate itself and give you an error.

N_L

Posted 27 January 2011 - 11:59 PM

The lamp energy is a measure of the amount or "strength" of the light coming out of the light bulb in the machine. What you perceive as white light is actually made up of lots of different wavelengths, some stronger and some weaker in output (see: Sodium bulb emission spectrum. As the bulb gets older, the bulb produces less light, some of which happens to be the at the 405 nm wavelength. The machine calibrates the amount of light it receives from the bulb and uses this as a basis for measurement... if too little light is received, it can't calibrate itself and give you an error.


Thanks for concise explanation.