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A dot-blot based assay to quantify collagen from large arteries of Physiology, Universidad Autonoma de Madrid; 2Vascular Surgery Unit, Hospital Moncloa, Madrid, Spain, 3Department of Human Anatomy, Universidad Autonoma de Nuevo León, Mexico

INTRODUCTION

Effect of Sirius Red concentration on dot intensity and background

Image Analysis

Collagen is a protein which provides tissues with strenght and resistance to stretch and contributes to fibrosis in several diseases when it is synthesized in excess. Therefore collagen quantification is important and

1.00 20 min

20 min

several methods are available, being colorimetric methods the most widely used:

Relative Integrated density

30 min

analysis, i.e. HP assay is timing consuming. 2. Sircol Collagen assay (SCA), based on the detection of Sirius Red (SR), which binds to collagen. The

Gelatin

Background

1.000

based on Dot blot and SR staining able to quantify intact and partially hydrolyzed collagen in arteries.

40 min

Densitometric parameter: integrated density (ID)

Sample loading

Water immersion for 2 min

0.95 0.94 0.93

0.90 0.0

0.1

0.975

0.1 0.2 0.3 0.4 0.5

0.950

RESULTS

0.1

0.2

0.3

Gelatin quantity ( g)

0.5

0.4

0.5

0.1

0.2

0.3

0.4

0.5

Protein quantity ( g)

Quantification of hydrolized collagen from rat carotid arteries

Effect of Sirius Red concentration on dot intensity and background

1 µL of sample or standard

0.2 0.3 0.4

0.925

Gelatin quantity (µg)

Optimization of a dot-blot based assay Experimental protocol

0.96

Gelatin

0.91

Dot blot is a commonly used techinque to quantify small quantities of proteins bound on membranes. The aim of the present study was to develop a selective, sensitive, high-throughput and cheap densitometric assay

0.97

BSA

0.92

main disadvantage is the interference with albumin and the lack of detection of partially hydrolized samples.

Wet filter paper

BSA

40 min

0.98

disadvantage is that release of HP requires acid or alkaline hydrolysis and neutralization before the sample

Membrane preparation

30 min

0.99

1. Hydroxyproline (HP) assay, based on the detection of HP, an imino acid unique to collagen. The main

METHODS

Interference of albumin with collagen quantification

Quantity (µg)

1Department

Relative integrated density

Program LB201 Abstract N° 9524

Luis Condezo-Hoyos1, Gabriel España-Caparros2, Angel L. Lopez de Pablo1, Pilar Rodríguez- Rodríguez1, Perla Y. Gutiérrez-Arzapalo1, Jesús Ancer3, M. Carmen Gonzalez1, Silvia M. Arribas1

0.0025% w/v

0.00125% w/v

0.000625% w/v

Sample

Mean integrated density

Gelatin (µg)

Collagen (mg gelatin/100 g dry weight)

1 2 3 4 5

3.516 3.523 3.528 3.536 3.533

0.4084 0.3882 0.3738 0.3508 0.3594

16.1 15.4 14.8 13.9 14.2

6 7 8 9 10

3.535 3.542 3.544 3.524 3.537

0.3537 0.3335 0.3278 0.3854 0.3479

12.0 11.3 11.1 13.1 11.8

11 12 13 14 15

3.553 3.546 3.558 3.539 3.555

0.3018 0.3220 0.2874 0.3422 0.2961

12.3 13.2 11.7 14.0 12.1

16 17 18 19 20

3.533 3.525 3.538 3.524 3.542

0.3594 0.3825 0.3450 0.3854 0.335

11.3 12.0 10.8 12.1 10.5

Gelatin standards

PVDF membrane

Methanol immersion for 1 min Membrane drying for 5 min at room temperature 0.1

Scanning Membrane drying on wet filter paper for 5 min at 37°C TCA 10% w/v in acetone washing for 10 min at RT, minimum speed Movil Rod

Image analysis (Image J)

0.2 0.3 0.4

0.5

0.1

0.2 0.3 0.4

0.5

Gelatin quantity (µg)

0.1

0.2 0.3 0.4

0.5

Effect of the incubation temperature on dot intensity and background 30°C

4°C

35°C

Sirius Red (0.0025% w/v) incubation for 30 min at 4°C, minimum speed Movil Rod

Water washing (three times)

Integrated density

25

3.68 3.66 3.64 3.62 3.60 3.58 3.56 3.54 3.52 3.50 3.48 3.46

y = -0.3472x + 3.6578 R² = 0.986

0

0.2 0.4 Gelatin (µg)

0.6

Samples

CONCLUSION 0.1

0.2 0.3 0.4

0.5

0.1

0.2 0.3 0.4

0.5

Gelatin quantity (µg)

0.1

0.2 0.3 0.4

0.5

We have developed a quick, simple, cheap and specific method to quantify collagen in vascular samples compatible with elastin purification. FUNDING: MICINN (FEM2009-13434-C02)

A Dot Blot  

A Dot Blot

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