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Agilent Protein 80 Kit 操作说明

Agilent Protein80Kit

Agilent Protein 80 Kit

Quick Start Guide

Protein 80 Kit (reorder number 5067-1515)

Protein Chips Protein 80 Reagents (reagent reorder number 5067-1516) & Supplies

25 Protein Chips z (red) Protein 80 Gel-Matrix (4 vials) in box labelled Part I. Store at 4°C

1 Electrode Cleaner z (blue) Protein 80 Dye Concentrate* in box labelled Part I. Store at 4°C

(white) Protein 80 Sample Buffer (4 vials) in box labelled Part II. Store at -20°C

Syringe Kit z(yellow)Protein 80 Ladder in box labelled Part II. Store at -20°C

1 Syringe 4 Spin Filters

*) “This product is provided under an agreement between Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corporation) and Agilent Technologies. The manufacture, use, sale or import of this product may be subject to one or more of U.S. patents, pending applications, and corresponding international equivalents, owned by Molecular Probes, Inc. The purchaser has the non-transferable right to use the product to detect protein and/or nucleic acids in microfluidics analysis systems for one or more of the subfields of research, development, quality control, forensics, environmental analysis, biodefense, food safety testing, veterinary diagnostics, or human diagnostics, according to use indicated on the product label or accompanying product literature. For information on obtaining a license, contact Molecular Probes, Inc., Business Development, 29851 Willow Creek Road, Eugene, OR 97402-9132. Tel: (541) 465-8300. Fax: (541) 335-0354.”

Assay Principles

Agilent Protein kits contain chips and reagents designed for sizing and analysis of proteins. Each chip contains an interconnected set of gel filled microchannels that sieves proteins by size as they are driven through it by means of electrophoresis. Agilent Protein kits are designed for use with the Agilent 2100 bioanalyzer only.

Assay Kits

The Agilent Protein 80 kit is designed for the sizing and analysis of proteins from 5-80 kDa and can be used to analyze cell lysates, column fractions or purified proteins.The complete Protein 80 Kit Guide can be found in the online help of the 2100 expert software.

Other protein kits from Agilent:

Protein 230 kit (reorder number 5067-1517)

Storage Conditions

?Keep all reagents in box labelled Part I refrigerated at 4°C when not in use to avoid poor results caused by reagent decomposition.

?Store Protein 80 Sample Buffer and Ladder (box Part II) at -20°C upon arrival. To avoid freeze-thaw cycles make aliquots depending on your daily use (e.g. 6 μl for ladder). The aliquot in use should be stored at 4°C.

?Protect all reagents from light. Remove light covers only when pipetting. The reagents contain dye that decomposes when exposed to light.

Agilent Protein 80 Kit Quick Start Guide

Equipment Supplied with the Agilent 2100 Bioanalyzer Additional Material Required (Not Supplied)

Setting up the Chip Priming Station

1Replace the syringe:

a Unscrew the old syringe from the lid of the chip priming station.

b Release the old syringe from the clip. Discard the old syringe.

c Remove the plastic cap of the new syringe an

d insert it into th

e clip.

d Slid

e it into the hole o

f the luer lock adapter and screw it tightly to the chip

priming station.

2Adjust the base-plate:

a Open the chip priming station by pulling the latch.

b Using a screwdriver, open the screw at the underside of the base plate.

c Lift the base plate an

d insert it again in position A. Retighten th

e screw.

?Chip priming station (reorder number 5065-4401)?Pipettes (10 μl, 20 μl, 100 μl, and 1000 μl) with compatible tips ?0.5 ml microcentrifuge vials ?Deionized water ?1M Dithiothreitol (DTT) solution or 2-Mercaptoethanol (BME)?Microcentrifuge

?Heating block or water bath for 0.5 ml vials

Physical Specifications Analytical Specifications Type

Specification

Type

Agilent Protein 80 Assay

Analysis run time

30minutes

Sizing range

5-80 kDa Number of samples 10samples/chip Typical sizing resolution 10%

Sample volume 4μl

Typical sizing accuracy 10% CV (CAII, BLG) Kit stability

4 months (Storage Temperature see individual box)

Sizing reproducibility

3% CV (CAII, BLG)

Sensitivity (Signal/Noise>3) 6 ng/μl CAII (15 ng/μl BSA) in PBS, 10 ng/μl

(CAII) in 0.5M NaCl (30ng/μl BSA in 0.5 M NaCl)CAII = Carbonic Anhydrase Quantitative range 60-2000 ng/μl CAII in PBS BSA = Bovine Serum Albumin Qualitative range 6-4000 ng/μl CAII and BLG BL = beta-Lactoglobulin

Quantitation reproducibility 20% CV (CAII, BLG) Compatible buffers see List of Compatible Buffers and Buffer

Compounds in your Protein 80 Kit Guide

Agilent Protein 80 Kit Quick Start Guide 3Adjust the syringe clip:

a Release the lever of the clip and lift it up or down to adjust it to the middle position.

Essential Measurement Practices

?Handle and store all reagents according to the instructions on the label of the individual box .

?Avoid sources of dust or other contaminants. Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results.?Upon arrival make aliquots of sample buffer and ladder with the typical amount required for daily use and store them at -20 °C. Keep the vial in use at 4 °C to avoid freeze-thaw cycles.?Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use.

?Protect all reagents from light. Remove light covers only when pipetting. The dye contained in the reagents decomposes when exposed to light and this reduces the signal intensity.?Always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results. ?Use a new syringe and electrode cleaners with each new kit.

?Use loaded chips within 5 minutes. Reagents might evaporate, leading to poor results.

?Do not touch the Agilent 2100 bioanalyzer during analysis and never place it on a vibrating surface.?Use 0.5 ml vials to denature samples. Using larger vials may lead to poor results, caused by evaporation.Agilent Protein 80 Assay Protocol - Edition April 2007

Handling DMSO

Some solutions may contain DMSO/dye. Because the dye binds to nucleic acids, it should be treated as a potential mutagen and used with appropriate care.

? Wear hand and eye protection and follow good laboratory practices when preparing and handling reagents and samples.

? Handle the DMSO/dye solutions with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.

Preparing the Gel-Dye Mix

1Transfer the content (650 μl) of an Agilent Protein 80 Gel matrix vial (z red) to a spin filter. Make sure the complete volume of 650 μl has been transfered.2Centrifuge at 2500g ± 20% for 15min.

3To the filtered and centrifuged Gel add 25 μl of the well vortexed Dye concentrate (z blue).

4Mix thoroughly for 10-20 s (Vortexer) until an uniform color is obtained.5

Label with the date and G/D (Gel/Dye). Use within 4 weeks.

WARNING

!

650 μl gel

25 μl dye gel-dye mix

Agilent Protein 80 Kit Quick Start Guide

Agilent Protein 80 Assay Protocol - Edition April 2007

Destaining Solution

1Transfer the content (650 μl) of another Protein 80 Gel matrix vial (z red) to a spin filter. Make sure the complete volume of 650 μl has been transferred.2Centrifuge at 2500g ± 20% for 15min.

3Label with the date and DS (Destaining Solution). Use within kit life time.

Preparing the Denaturing Solution

1For reducing conditions, add 3.5 Vol-% of 1 M Dithiothreitol (DTT) or

?-mercaptoethanol (BME) to an aliquot of sample buffer (e.g. 1.0 μl DTT or BME to an aliquot of 28.6 μl Sample Buffer). Alternatively, for non-reducing conditions add 3.5 Vol-% of water to your aliquoted sample buffer vial.2Vortex for 5s.

Preparing the Samples and the Ladder

1Combine 4μl protein sample and 2μl denaturing solution in a 0.5ml vial.

2Place sample vial and a vial containing a 6 μl aliquot of Protein 80 Ladder (z yellow) at 95 oC for 5min. Cool down afterwards.3Spin tubes for 15s.

4Add 84μl deionized water to samples and ladder and vortex.

Loading the Gel-Dye Mix and the Destaining Solution

1Adjust the base-plate of the chip priming station to position A and the syringe clip to its middle position.

2

Take a new protein chip out of the sealed bag and put it on the chip priming station.3Pipette 12μl of gel-dye mix in the well marked .4Put plunger at 1 ml and close chip priming station.

5Press plunger until held by clip, wait 60s, then release clip.6Wait for 5 s. Slowly pull back plunger to 1ml position.7Pipette 12μl of gel-dye mix in all wells labeled with “G”.8Pipette 12μl of destaining solution in well .Loading the Ladder and the Samples

1Pipette 6μl of sample in all 10sample wells (Note: all 10 sample wells must be filled either with ladder or sample).

2Pipette 6μl of the prepared ladder in the well marked

.

3Place the chip in the Agilent 2100 bioanalyzer and start the assay immediately.

Technical Support In the U.S./Canada: 1-800-227-9770 (toll free); lsca-ibs-support@https://www.sodocs.net/doc/4515616154.html,. In Europe: call your local Customer Care Center; bio_solutions@https://www.sodocs.net/doc/4515616154.html,. In Japan: 0120 477 111; yan_ccr@https://www.sodocs.net/doc/4515616154.html,. In Asia Pacific: call your local Customer Care Center; Bioanalyzer_ap@https://www.sodocs.net/doc/4515616154.html,

Further Information Visit Agilent Technologies’ unique Lab-on-a-Chip web site. It is offering useful information, support and current developments about the products and the technology: https://www.sodocs.net/doc/4515616154.html,/chem/labonachip .

destaining solution 650 μl gel

denaturing e.g. 1.0 μl DTT solution +28.6 μl sample

buffer

4 μl sample

2 μl denaturing solution 84 μl deionized

water

12μl gel-dye Pressurize

12μl gel-dye 12μl destain

.6μl sample .6μl ladder

*G2938-90063*Part Number: G2938-90063

? Agilent Technologies, Inc. 2000, 2001-2007

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