[HusamEddin]

Hello there
It's been a long time since i've posted my last post in this awesome forum
wanna ask,
Why do we use distilled water (in addition to the reagent) as a blank in spectrophotometry in some trials (like determining the con. of total protein in serum) while still don't in others (like glucose in serum) ?
first of all , why do we use the distilled water to zero the device ? is it because it has ions that may contribute to the absorbance which are also may be found in the serum itself ?

wish you the best

[Yggdrasil]

In general, you would want to use the buffer your protein is as a blank in spectrophotometry since some buffer components do will contribute to absorbance.  However, if you find that a solution blanked with buffer and a buffer blanked with distilled water behave identically, you can just blank with distilled water in the future as this is more convenient.

[Adenosine TriBossphate]

I know I'm currently doing research in measuring ATPase activity with time trials to measure the phosphate hydrolyzed by a cell sample. The dye we add to the solution will turn green in the presence of phosphate (more phosphate hydrolyzed the more green). To account for the initial 'green' the dye adds, we add the dye to DI water (which shouldn't have phosphate in it) in order to zero the device. Depending on what sort of analysis you're doing, it should be for similar reasoning as this.

[HusamEddin]

I'm sorry , I didn't see the first reply , I'll read it again
(this reply has been modified and no need for it , if you can delete it please)

[HusamEddin]

In general, you would want to use the buffer your protein is as a blank in spectrophotometry since some buffer components do will contribute to absorbance.  However, if you find that a solution blanked with buffer and a buffer blanked with distilled water behave identically, you can just blank with distilled water in the future as this is more convenient.

here's what i've understood from you:
I'm right now determining the concentration of "albumin" in serum
Albumin acts as a buffer,
So, I have to record its absorbance at the same time it acts as a buffer , because its buffer components contribute to the absorbance
and so, I have to add it not only to the sample cuvette and calibirator , but also to the blank (in addition to the reagent)
but because adding it with distilled water by side , will give the same result , then it is convenient to use only the distilled water instead.

Am I right with that ??

[Yggdrasil]

When I talk about buffer components, I'm talking about components in solutions that biochemists typically make (e.g. phosphate buffered saline [PBS]).  For example, if you are performing a Bradford assay for a purified protein in PBS, your would ideally blank the spec with a sample of Bradford reagent + PBS.   

Since you're measuring the concentration of biomolecule in serum, it's not clear what the ideal blank is for your measurements since it will be difficult to obtain a sample of serum lacking the particular component you want to measure.  Whether distilled water or some other solution will be a suitable blank depends on the exact details of the method you are using.

[HusamEddin]

Sorry for being late to reply , I had a "Nutrition" exam.
back to the question
"Whether distilled water or some other solution will be a suitable blank depends on the exact details of the method you are using"
really i didn't got this one

but I'll tell you what method I'm using , to determine if distilled water is suitable for my trial or not

be back after minutes

[HusamEddin]

Quantitative determination of Albuminin human serum by enzymatic method.
Albumin
Albumin is bound by the BCG dye (Bromocresol Green) to produce and increase in the blue green color in a pH 3.8 acid medium.
The color increase is proportional to the concentration of albumin present in the sample.
Albumin determination is useful in diagnosis of hepatic and renal pathologies.
Procedure
Wave length: 546 nm (530-550)
Temperature: +25/30/37 C
Pipetting in tubes:

Blank
   Standard
    Sample

Reagent (R2)   

1000  μl
   1000  μl
    1000  μl

Distilled water  10 μl

Standard   

10 μl

Sample

10 μl

Cont.
Mix, incubate for 5 min at 37 C or 10 min at room temperature (+15-25C) and read the absorbance of standard sample tubes.
Color is stable at least 60 min at room temperature.
Calculation:
Albumin g/dl = (A) Sample/ (A) Standard * 4.0

Serum: 3.5 – 5.3 g/dl

[HusamEddin]

this is it , the method , so why adding the distilled water ?

[Yggdrasil]

How are you preparing your standard?

[HusamEddin]

prepared already
It's a bottel
I have a kit that contains some tools , and among them is a bottel that contains prepared standard
I just add some of it to the calibrator cuvette and add some of my serum to it , according to the table I've wriiten before
and by the way this table is written on a paper comes with the other tools in that kitt
this paper who tells me to add the distilled water to the blank in addition to the reagent (which comes also in a bottel among the other contents)
I just wanted to know why this distilled water ?

[Yggdrasil]

Do you know what diluent your albumen standard is dissolved in?

[HusamEddin]

no , but if it would be written on the paper glued on the bottel, then I'll take a pic of it by my phone's camera next sunday :/

[Yggdrasil]

Basically, the purpose of the blank is to provide a solution without albumin so that you can measure the absorbance in the absence of albumin to provide a baseline for the rest of the measurements.  Ideally, the blank should be the diluent in which the albumin standard is dissolved (for example, distilled water is an appropriate blank if the albumin is dissolved in distilled water).

One problem with this blank, however, is that it will not account for any factors present in the serum which might interfere with the assay.  Using the absobance values obtained from the standard to estimate the concentration of albumin in serum requires the assumption that albumin is the only substance present in the serum that will change the absorbance of the solution.  In general, this is not a safe assumption to make, but since this method is fairly widely used, I would think that others have tested this assumption and found that it is for the most part true.

[HusamEddin]

I'm sorry I took a pic for the table of the reagent contents :/
next time I'll take the right pic
..
about your last reply , if this is the case , then why did we use the distilled water in a trial measures the albumin in serum , but didn't use it in a trial measures the glucose in serum ??
why that difference ?