For the rapid, sensitive and accurate measurement of Ferrous and/or Ferric ions in various samples.

Transcription

1 ab83366 Iron Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Ferrous and/or Ferric ions in various samples. This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Overview 3 2. Protocol Summary 3 3. Components and Storage 4 4. Assay Protocol 5 5. Data Analysis 6 6. Troubleshooting 8 2

4 1. Overview Iron is essential to nearly all known organisms. It is generally stored in the centre of metalloproteins, in the heme complex, and in oxygen carrier proteins. Inorganic iron also contributes to redox reactions in the iron-sulfur clusters of many enzymes, such as nitrogenase and hydrogenase. Abcam's Iron Assay Kit provides a simple convenient means of measuring Ferrous and/or Ferric ions in samples. In the assay, ferric carrier protein will dissociate ferric into solution in the presence of acid buffer. After reduction to the ferrous form (Fe 2+ ), iron reacts with Ferene S to produce a stable colored complex and give absorbance at 593 nm. A specific chelate chemical is included in the buffer to block copper ion (Cu 2+ ) interference. The kit measures iron in the linear range of 0.4 to 20 nmol in 50 µl sample, or 8 µm to 400 µm iron concentration in various samples 2. Protocol Summary Standard Curve Preparation Sample Preparation Add Iron Probe Measure Optical Density 3

5 3. Components and Storage A. Kit Components Item Quantity Iron Assay Buffer 15 ml Iron Probe 12 ml Iron Reducer 0.7 ml Iron Standard (100 mm) 0.1 ml * Store the kit at -20 C, protect from light. Warm Assay Buffer to room temperature before use. Briefly centrifuge vials prior to opening. Read the entire protocol before performing the assay. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 4

6 4. Assay Protocol 1. Standard Curve Preparation: Dilute 10 µl of the 100 mm Iron Standard with 990 µl dh 2 0 to generate 1 mm standard Iron. Add 0, 2, 4, 6, 8, and 10 µl of the diluted Iron standard into a 96-well plate to generate 0, 2, 4, 6, 8, and 10 nmol/well standard. Bring the volume to 100 µl with Assay Buffer. Add 5 µl iron reducer to each standard well. 2. Sample Preparation: Samples can be tested for ferrous (Fe 2+ ), or total Fe(II+III) or ferric (Fe 3+ ) ion. a. For liquid samples: Liquid sample can be tested directly. Normal serum Iron ~10-40 µm. b. For tissue or cell samples: Tissue or cells can be lysed in 4-10 volume of Iron Assay Buffer, centrifuge at x g for 10 min to remove insoluble materials. We suggest testing several doses of your samples to make sure the readings are within the standard curve range. a. For the Iron (II) assay: Add 1-50 µl samples to sample wells in a 96-well plate and bring the volume to 100 µl/well with Assay Buffer. Add 5 µl Assay Buffer to each sample without Iron Reducer. 5

7 b. For total Iron (II+III) assay: Add 1-50 µl samples to sample wells in a 96-well plate and bring the volume to 100 µl/well with Assay Buffer. Add 5 µl Iron Reducer to each sample to reduce Iron (III) to Iron (II). 3. Incubate iron standards and samples for 30 min at 25 C. 4. Add 100 µl Iron Probe to each well containing the iron standard and test samples. Mix well. Incubate the reaction for 60 min at 25 C, protect from light. 5. Measure the OD at 593 nm in a microplate reader. 5. Data Analysis Subtract zero standard reading from all standard and sample readings. Plot iron standard curve. Apply sample readings to the standard curve. Iron (II) and total iron (II+III) contents of the test samples can then be acquired directly from the standard curve. Iron (III) content of the test sample can be calculated as: total iron (II+III) iron (II) The iron(ii), iron(iii), and total iron(ii+iii) concentration in the samples can be calculated: Concentration = Sa / Sv (nmol/µl, or mm) 6

8 Where: Sa is the iron (II), iron (III), or total iron (II+III) content of unknown samples (in nmol) from standard curve. Sv is sample volume (µl) added into the assay wells. Iron ion molecular weight is g/mol. 7

9 6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 8

10 Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 9

11 Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by (technical@abcam.com) or phone (select contact us on for the phone number for your region). 10

12 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. 11 All information / detail is correct at time of going to print.