Rat PAI-1                                       Price : 550 €

For Research Use Only

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Plasminogen Activator Inhibitor-1 (PAI-1) is a glycoprotein and member of the

serine proteinase inhibitor (serpin) superfamily. PAI-1 is the primary inhibitor

of tissue-type plasminogen activator (tPA) and the urokinase-type plasminogen

activator (uPA). This inhibition exhibits antiproteolytic properties that can

lead to myocardial infarction, thromboembolic disease with elevated levels of

PAI-1. Additionally, PAI-1 is thought to play a role in the function of tissue

remodeling and tumor metastasis.

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This is an ELISA (Enzyme-Linked Immunosorbent Assay) for the quantitative analysis of

PAI-1 levels in biological fluid. This test kit operates on the basis of sandwich ELISA

where active PAI-1 complexes with uPA and is quantitfied with the use of an HRP labeled

secondary antibody.

The functional or active PAI-1 binds to the uPA coated on the well of the microtiter plate.

The latent and complexed forms of PAI-1 will not bind and are discarded at a later washing

step. Next, PAI-1 primary antibody is added to the wells binding to the captured PAI-1 on

the microtiter plate. HRP conjugated secondary antibody is then added for detection of the

active PAI-1. Quantitative test results are obtained by the measure and comparison of the

sample and standard absorbance readings.

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1. Rat PAI-1 Standard: 1 vial.

2. Substrate: 10 mL. Stabilized 3,3', 5,5' Tetramethylbenzidine (TMB) plus

3. Anti-PAI-1 Primary Antibody: 1 vial of anti-rat PAI-1 antibody.

4. HRP Secondary Antibody: 1 vial of HRP conjugated secondary antibody.

5. Coated Plate: A 96 well microplate with PAI-1 capture antibody precoated on each

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2. TBS buffer (see Reagent Preparation).

3. Blocking buffer (see Reagent Preparation).

4. Wash buffer (see Reagent Preparation).

5. DI water.

6. Microplate reader with 450 nm filter.

7. Microplate shaker with uniform horizontal circular movement up to 300 rpm.

8. Beakers, flasks, cylinders, etc. required for preparation of reagents.

10. Plastic film or plate cover to cover plate during incubation.

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components of any other kit. This kit is designed to work properly as provided.

the bottle (if your tip is unclean you could contaminate your substrate).

5. All specimens should be considered potentially infectious. Exercise proper handling

precautions.

7. Use aseptic technique when opening and removing reagents from vials and bottles.

8. Keep plate covered except when adding reagents, washing or reading.

9. Kit components should be stored as instructed when not in use.

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1. Always use new pipette tips for the buffer, conjugate, standards, samples etc.

2. Before pipetting a reagent, rinse the pipette tip three times with that reagent (i.e. fill the

tip with the desired amount of reagent and dispense back into the same vial - repeat 2

times). Now the tip is properly rinsed and ready to dispense the reagent into your well

or test tube.

3. When pipetting into the wells, DO NOT allow the pipette tip to touch the inside of the

well, or any of the reagents already in the well - this can cause cross contamination.

4. Standards and samples should be assayed in duplicate.

5. To quantitate, always run a standard curve when testing samples.

6. Gently mix specimens and reagents before use. Avoid vigorous agitation.

7. Before taking an absorbance reading, wipe the outside bottom of the wells with a lintfree

wiper to remove dust and fingerprints.

8. Desiccant bag must remain in foil pouch with unused strips. Keep pouch sealed when

not in use to maintain a dry environment. Seal with a heat sealer. If a heat sealer is not

available, thoroughly close the open end with tape. Remove excess air before sealing.

9. If not using the entire plate at once, then prepare only the appropriate amount of

standard and primary antibody. The remaining stock solutions should then be refrozen

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(DiaPharma) collection media. Collection should be in accordance with the collection vials

manufacturers instructions or in a 1:10 ratio of collection media to blood.

Immediately, upon collection of blood, the samples should be centrifuged at 3000 x g. This

should ensure the removal of platelets as they can release PAI-1 that in turn complexes with

uPA. The plasma can be transferred to a clean plastic tube and stored frozen for up to one

Note: Detergents such as Triton X cause interference with the assay. If using detergent

extracted samples, it is necessary to dialyze the samples overnight to remove the detergent.

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The following solutions should be prepared fresh before starting the assay.

-TBS Buffer: 0.10 M TRIS, 0.15 M NaCl, pH 7.4

-Blocking Buffer: 3% BSA in TBS buffer.

-Wash Buffer: 0.05% Tween, 0.1 % BSA in TBS buffer.

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Note: This assay should be performed at room temperature.

1. Remove microplate from the bag.

2. Prepare standards as indicated in the provided dilution table.

Note: The standards should be applied to the plate immediately upon preparation.

Scheme I for suggested template design.

4. Shake the plate at 300 rpm on a plate shaker for 30 minutes.

5. Wash wells according to the following wash procedure:

a. Remove contents of the plate by inversion into an appropriate disposal device.

b. Tap out remaining contents of the plate onto a lint free paper towel.

d. Let stand for 2-3 minutes.

e. Repeat procedure 2 more times then proceed to step "f".

f. Remove contents of the plate by inversion into an appropriate disposal device.

g. Tap out the remaining contents of the plate onto a lint free paper towel then proceed

to step 6.

Note: The decanted wells should be void of visible moisture before proceeding. If

moisture is still visible then follow step "g" until satisfactory results are obtained.

6. Add entire vial of primary antibody to 10 mL of the 3% BSA blocking buffer to make a

working concentation.

8. Shake plate at 300 rpm on the plate shaker for 30 minutes.

9. Wash wells according to step 5 located above in this section.

12. Shake plate at 300 rpm on the plate shaker for 30 minutes.

13. Wash wells according to step 5 located above in this section.

16. If accounting for substrate background, use 2 to 8 wells as blanks with only substrate

absorbance values of the wells being assayed.

NOTE: Some microplate readers can be programmed to do these subtractions

automatically when reading the plate. Consult your instrument manual.

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1. Subtract the average O.D. value of the blank wells (BLK) from all other pairs of wells.

2. Average the O.D. values for each pair of duplicate wells.

3. Plot a standard curve using the average O.D. value for each standard value versus the

concentration of standard.

4. Determine the concentration of each unknown by interpolation from the standard

curve.

1 2 3 4 5 6 7 8 9 10 11 12

A S1 S2 S3 S4 S5 S6 S7 S8 U1 U2 U3 U4

B S1 S2 S3 S4 S5 S6 S7 S8 U1 U2 U3 U4

C U5 U6 U7 U8 U9 U10 U11 U12 U13 U14 U15 U16

D U5 U6 U7 U8 U9 U10 U11 U12 U13 U14 U15 U16

E U17 U18 U19 U20 U21 U22 U23 U24 U25 U26 U27 U28

F U17 U18 U19 U20 U21 U22 U23 U24 U25 U26 U27 U28

G U29 U30 U31 U32 U33 U34 U35 U36 U37 U38 U39 BLK

H U29 U30 U31 U32 U33 U34 U35 U36 U37 U38 U39 BLK

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Assay Range: 0.0- 50 ng/mL

Samples with uPA levels higher than 50 ng/mL should be diluted in similar media devoid of

active uPA.

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1. Declerck, P.J., et al. J Intern Med (1994) 235:4250432

2. Winman, B., et al. Scand J Clin Lab Invest Suppl (1999) 230:23-31

3. Winman, B., et al. Thomb Haemostas (1984) 5-1:R9-R17

4. Colucci, M., et al. J Clin Invest (1985) 75:818-824

5. Kruithof, E. K., et al. Blood (1987) 70:1645-1653

6. Ranby, M., et al. Fribrinolysis (1990) 4:54-55

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Oxford Biomedical Research, Inc. makes no guarantee of any kind, expressed or implied,

which extends beyond the description of the material in this ELISA kit, except that these

materials and this kit will meet our specifications at the time of delivery. Buyer's remedy and

Oxford Biomedical Research, Inc.'s sole liability hereunder is limited to, at Oxford

Biomedical Research, Inc.'s option, refund of the purchase price of, or the replacement of,

material that does not meet our specification. By acceptance of our products, Buyer

indemnifies and holds Oxford Biomedical Research, Inc. harmless against, assumes all

liability for the consequence of its use or misuse by the Buyer, its employees, or others. Said

refund or replacement is conditioned of Buyer notifying Oxford Biomedical Research, Inc.

within (30) days of the receipt of product. Failure of Buyer to give said notice within said

thirty (30) days shall constitute a waiver by the Buyer of all claims hereunder with respect to

said material(s).