Protein Carbonyl Content Assay Kit

$150.00

  • Model: AS012
  • 50 Units in Stock
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Protein Carbonyl Content Assay Kit

Catalogue No.: AS012

1.  Overview

Protein carbonyl groups are an important and immediate biomarker of oxidative stress. DNPH tagging of protein carbonyls has been one of the most common measures of oxidative stress. DNP hydrazones formed from the reaction are easily quantifiable at 375 nm or 370 nm.

K012 Protein Carbonyl Content Assay Kit is designed to provide a simple and accurate method of

quantifying carbonyls in protein samples. Using BSA as an example, a 1 mg (~15 nmol) sample has a

detection limit of about 0.15 nmol carbonyl, where BSA typically contains approximately 1-3 nmol

carbonyl/mg.

 

2.  Protocol Summary

 

Sample Preparation

Removal of Nucleic acids

Standard Curve Preparation

Add Reaction Mix

Measure Optical Density

 

 

 

 

 

 

 

 

 

3.  Components and Storage

A.  Kit Components

Item

Quantity

 

 

DNPH Solution

11 mL

100% TCA Solution

3 mL

10% Streptozocin Solution

1 mL

6 M Guanidine Solution

20 mL

96-Well Clear Plate

1 each

 

 

Store the kit at +4°C and protect from light. Please read the entire protocol before performing the assay. Avoid repeated freeze/thaw cycles.

We suggest the use 1.5 ml microcentrifuge tubes for all reactions, since they are very convenient for all processing steps.

REAGENTS: Place 10 ml acetone (not provided) in freezer (-20°C) prior to starting the following

procedure.

DNPH, TCA, STREPOZOCIN, GUANIDINE: All solutions are ready to use as supplied. Store at 4°C in the dark. Warm the DNPH, Streptozocin and Guanidine to room temperature before use. Keep TCA on ice.

B.  Additional Materials Required

MICRO CENTRIFUGE

PROTEIN ASSAY REAGENTS

ACETONE

PIPETTES AND PIPETTE TIPS

MICROPLATE READER

96 WELL PLATE

ORBITAL SHAKER

4. Assay Protocol

A. DNPH Assay

1. Sample Preparation:

Dissolve  samples  in  dH2O  and  centrifuge  to  spin  down  any insolubles.  Dilute  samples  with  dH2O  to  approx.  10  mg/ml protein. If the protein is very dilute, it can be concentrated.(PEG dialysis bag OD≤10KD.) Use 100 μl of sample containing approximately  0.5-2  mg  protein  per  assay.  Include  a  reagent background control by using 100 μl of dH2O alone.

Note:

Nucleic  acids  interfere  with  the  assay.  Samples  containing significant nucleic acid should be treated with Streptozocin (10 μl per 100 μl sample). Leave for 15 min at room temperature, spin

at maximum speed  for 5 min and  transfer supernatant  to a new tube. Check 280/260 nm ratio to make sure it is greater than 1.

2. Add  100  μl  DNPH  to  each  sample,  vortex  and  incubate  for 10 min at room temperature.

3. Add 30 μl of TCA to each sample, vortex, place on ice for 5 min, spin  at  maximum  speed  for  2  min,  remove  and  discard supernatant without disturbing pellet.

4. Add  500  μl  of  cold  acetone  to  each  tube  and wash  the  pellet. 30 seconds  in  a  sonicating  bath  is  typically  sufficient  to effectively  disperse  the  pellets.  Place at -20°C  for  5 min  then centrifuge for 2 min and carefully remove the acetone.

Caution: The acetone pellet  is much more easily disturbed  than  the TCA pellet. Repeat the acetone wash step once more to remove free DNPH.

5. Add  200  μl  of  Guanidine  solution  and  sonicate  briefly.  Most proteins will be resolubilized easily at this point. If your protein is resistant  to  resolubilization  sonicate  for  a  few  seconds  then  let the solution sit at 60°C  for 15-30 min. Spin very briefly  to pellet any unsolubilized material and transfer 100 μl of each sample to the 96-well plate.

Note: You must use the 96-Well plate included for accurate calculation of carbonyl content.

6. Measure OD at ~375 nm or 370 nm in a microplate reader.

B. Protein Assay:

Transfer 5 μl of each sample to another set of wells and perform a protein assay to precisely determine the amount of protein per sample (use BSA as the standard protein when generating your standard curve).

Notes:

a) If you are using more than 1 mg protein per sample, it must be diluted so that no more than 25 μg protein is used in the protein assay. It is Important to correct for any sample losses.

b) The  BCA  Protein  Quantification  Assay  (FN-K001)  shows minimal  interference.  The  Bradford 

protein  assay  is inappropriate for this purpose since guanidine interferes.

5. Data Analysis

Correct  background  by  subtracting  the  value  derived  from  the reagent  background  control  from all  readings.  The background reading should not be very high but must be subtracted. Determine protein content of samples  from  protein  standard  curve.

Note:

The  BCA  Protein  Quantification  Assay  is  best  fit  by  a  2nd  order curve  rather  than a straight  line. Determine the carbonyl content as follows:

C = [(OD 375 nm)/6.364) x (100)] nmol/well

CP = nmol carbonyl per mg protein

= (C/P) x 1000 x D

Where:

6.364  is  the  extinction  coefficient  using  the  enclosed  96 well  plate in mM (= 22 mM-1 cm-1 x

0.2893 cm path length in well)

C is the Carbonyl in your sample well (nmol)

P is the protein from standard curve x 20 = μg/well

D is the dilution or concentration step applied to sample

1000 is the factor to convert μg to mg

 

Typical Standard Curve  

 

 

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