Antibody Array User`s Guide
Contents
1. By centrifugation place the slide in a 50 ml conical tube and close the cap Centrifuge the tube at 1300 x g for 5 10 minutes The slide is now ready for scanning Note If you do not have access to a microarray scanner you can send the slides to our lab for scanning Please visit our website for details To prepare the slides for shipping place the slides back in the slide holder Cover the slide holder with aluminum foil to protect the slides from light Send the package at room temperature Please include the array information and your contact information name organization phone and email address in the package Shipping address Attn Array Scanning Service Phone 408 737 1702 Full Moon BioSystems Inc Email support fullmoonbio com 754 North Pastoria Avenue Sunnyvale CA 94085 United States Page 15 Antibody Array User s Guide Full Moon BioSystems Inc ARRAY SCANNING AND IMAGE ANALYSIS All scanners that are compatible with 75mm x 25mm 3in x 1in microscope slides can be used to scan Full Moon BioSystems antibody arrays Recommended Scanning Resolution 20um or higher 10um 5um etc Compatible Systems Manufacturer Product Name Required Accessory Agilent Technologies SureScan Microarray Scanner All models Alpha Innotech AlphaScan Microarray Scanner NovaRay Detection Platform Applied Precision arrayWoRx e Biochip Reader Biomedical Photometrics The DNAscope HR Bio Rad VersArray Ch2. Add 1 8 g of Dry Milk to 60 ml of Blocking Reagent Shake to mix Be Solution sure the milk powder is completely dissolved Use within one week 3 Coupling Add 0 36 g of Dry Milk to 12 ml of Coupling Reagent Shake to mix Be Solution sure the milk powder is completely dissolved Use within one week 20 RXN Kit KAS20 1 1X Wash Make 1 10 dilution For example add 100 ml of 10X Wash Buffer to 900 Solution ml of ddH O to make 1L of 1X Wash Solution Shake to mix If you plan to perform 20 assays within one week add 18 g of Dry Milk to 2 Blocking 600 ml of Blocking Reagent Be sure the milk powder is completely Solution dissolved For two assays aliquot 60 ml of Blocking Reagent and add 1 8 g of Dry Milk Shake to mix Use within one week If you plan to perform 20 assays within one week add 3 6 g of Dry Milk to 3 Coupling 120 ml of Coupling Reagent Be sure the milk powder is completely Solution dissolved For two assays aliquot 12 ml of Coupling Reagent and add 0 36 g of Dry Milk Shake to mix Use within one week Rev 11 3 Page 8 Antibody Array User s Guide Full Moon BioSystems Inc PROTOCOL DETECTION BY CY3 STREPTAVIDIN IMPORTANT WARM REAGENTS BEFORE USE See Reagent Preparation for detailed instructions A Protein Extraction Note It is recommended that protein extraction is performed with the Extraction Buffer provided in the Antibody Array Assay Kit KAS02 Other protein extraction procedures or lysis b
3. at 750 x g for two minutes to remove excess fluid Blot excess drops from the bottom of the column Discard the wash tubes and the excess fluid Do not allow the gel material to dry excessively Process the samples within the next few minutes Transfer up to 100uL of protein extract by carefully dispensing the sample directly onto the center of the gel bed at the top of the column without disturbing the gel surface Do not touch the sides of the columns with the reaction mixture or the sample pipet tip since this can reduce the purification efficiency Place the column into a collection tube and place both together into the rotor Maintain proper column orientation Spin the column and collection tube at the 750 x g for 2 minutes The purified protein will collect at the bottom of the collection tube Discard the spin column Proceed immediately to the next step C Lysate quantification and QC Rev 11 3 1 Lysate quantification Measure lysate sample s protein concentration by UV absorbance spectroscopy A280 BCA assay Bradford assay or other quantification methods UV Absorbance A280 Measure protein sample s absorbance OD Use Labeling Buffer or water as blank Note The minimum absorbance is 3 OD If the OD for your sample is too low the sample must be concentrated at 4 C in a vacuum centrifuge such as SpeedVac or using YM 10 filters Millipore Corporation Note Only absorbance OD reading is required This
4. proceed directly to Step D Protein Labeling Important After centrifugation a thin milky film often collects at the top When removing the sample for labeling it is important that the milky film is not transferred to the new tube Make sure that the pipette tip pierces through the milky film then release the pipette to aspirate the clear yellowish serum plasma It s helpful to wipe off the pipette tip with a Kimwipe before releasing the sample in the new tube B Buffer Exchange Lysate Purification Rev 11 3 Page 10 Antibody Array User s Guide Full Moon BioSystems Inc Important This step ensures the removal of unwanted buffer from your protein extract and replaces it with the Labeling Buffer provided in the Antibody Array Assay Kit 11 The sample volume capacity of each spin column is 100 uL Gently tap the columns to ensure that the dry gel has settled to the bottom of the column Remove the top column cap and reconstitute the column by adding 650 uL of Labeling Buffer Replace the column cap and vortex vigorously for about 5 seconds Remove air bubbles by sharply tapping the bottom of the column Allow at least 30 to 60 minutes of room temperature hydration time before using the column Note If the column was stored at 4 C allow the column to reach to room temperature before use After hydration remove the top column cap and then remove the column end stoppers from the bottom Spin the column in its wash tube
5. reading will be used in later steps to determine the amount of sample used for labeling and coupling Conversion to protein concentration mg ml is not necessary Most A280 assays assume 1 OD 1 mg ml This Page 11 Antibody Array User s Guide Full Moon BioSystems Inc Rev 11 3 conversion is not accurate for lysate samples because the non protein components such as nucleic acids insoluble lysate factors absorb UV light and interfere with the assay BCA and Bradford assays either assay can be used to determine lysate sample s protein concentration The minimum protein concentration required is 2 mg ml Lysate Quality Control by A280 Assay Optional but highly recommended This step can be performed at the same as Step 1 above The quality of lysate sample directly affect assay results The lysate sample should be as clear and transparent as water Cloudy or unclear lysate sample will result in low labeling efficiency and high background A280 spectrum is a good way to determine whether the lysate sample has sufficient clarity Clear lysate produces two well separated peaks at 200 230nm and 240 280nm If the peaks are not well separated it indicates the lysate is not clear enough To improve the lysate s quality store the lysate at 70 C for 10 to 20 minutes Remove from the freezer and immediately centrifuge at 18 000 x g for 15 to 20 minutes at 4 C Save the top clear layer and discard the rest LI nis File Edit He
6. Antibody Array User s Guide Full Moon BioSystems Inc Antibody Microarray User s Guide FULL MOON BioSystems Rev 11 3 Page 1 Antibody Array User s Guide Full Moon BioSystems Inc TABLE OF CONTENTS INTOGUCHION 2 2802 inte teats aka mabaya ka date A ka a kaka Gini dential ees 3 Antibody Alaya u crates dale cpeadees thatch ate idle anes ebhnte ce eateeebeielel dd hidi die daltons 3 Antibody Array ASSAY Kitu uuu ayu aqu ates alerectantaseceiudadecttateaddcycsieeeteataneatsanaieeintaa eee Oa TRS 3 HOW IE WOKS irs sicceicis sects ancien atana aaae aa A aE A aaea aa aa aaa EEE aaa aa attendees 4 Experimental consideratiorISu uuu un tikonni aeaa eaa E ea pa Qusa qaqa waqaspa AKEKE 4 G ITIDOnST1S uuu ukasa a cadets wea aaa aaa qawa ataqa aqa Aa naaa a aa Ra a a a a a aSa Ga 5 Additional Materials REQUIrCO tccesissccadesszestectactdeissenectantasecevdntecte aana a aa aa aa Aa Raae 7 Reagent Preparation kssin aeina aa aa aa aiaa Balaka aE Ea aE Ea aaa aaa a EEA 8 Protocol uu kunu A AES RS 9 Array Scanner Recommendations U U L nn 16 Rev 11 3 Page 2 Antibody Array User s Guide Full Moon BioSystems Inc INTRODUCTION Antibody Microarray is a high throughput ELISA based platform for efficient protein expression profiling screening and comparison between normal diseased or treated samples Full Moon BioSystems antibody arrays allow researchers to detect and analyze hundreds of native protei
7. Only use PBS to wash cells To protect protein activity avoid using trypsin or other reagents 2 Vortex rigorously for 30 seconds to 1 minute Incubate on ice for 10 minutes Repeat five times Centrifuge the mixture at 10 000 x g for 5 minutes at 4 C Transfer all liquid to a clean tube Discard the beads Centrifuge the new tube with liquid at 18 000 x g for 15 20 minutes at 4 C The supernatant on top should look colorless and transparent as water Transfer the clear supernatant to a clean tube Important If the supernatant appears to be cloudy centrifuge again at 18 000 x g for 15 to 20 minutes at 4 C Check again If the supernatant is still not clear store the lysate at 9 Dr o p Rev 11 3 Page 9 Antibody Array User s Guide Full Moon BioSystems Inc 7 70 for 10 to 20 minutes Immediately centrifuge at 18 000 x g for 15 to 20 minutes at 4 after removal from freezer Save the top clear layer and discard the rest The supernatant should look colorless and transparent as water Proceed immediately to Step B Buffer Exchange Lysate Purification Tissues 1 ONO Wash tissues with ice cold 1X PBS with vortexing Remove and discard PBS Repeat 3 5 times Important If blood in the tissues gets in the lysate it will lead to high background on the arrays Be sure to remove blood from the tissues completely Increase the number of PBS washes if necessary When blood has been completely removed the tissues
8. ake for 10 seconds Discard the water 2 Repeat ten times Important It is critical to rinse the slide extensively to completely remove Blocking Solution from the slide surface A clean slide will ensure a uniform and low background Because antibodies are covalently immobilized on the slide surface rigorously washing will not strip them off After the last rinse cycle the thin layer of water left on the slide surface should appear uniformly smooth across the entire surface If it looks spotty it means the surface is not clean Please repeat Step 4 5 Shake off excessive water on the slide surface Proceed immediately to the next step Page 13 Antibody Array User s Guide Full Moon BioSystems Inc Note Do not allow the slide to dry out If you are not ready to start coupling place the slide back in the conical tube filled with clean water F Coupling Note You should prepare the Protein Coupling Mix Step F 1 in advance so that you can start coupling immediately after blocking In a tube add 6 ml of Coupling Solution See Reagent Preparation and one tube of biotinylated sample 80 150 OD or 30 80 ug Vortex briefly to mix Label it as Protein Coupling Mix Important Make sure Coupling Solution is at room temperature and that the dry milk is completely dissolved before use Place the slide in Well 1 or any clean well of the Coupling Chamber Slowly pour all 6 ml of Protein Coupling Mix over th
9. array and 4 Detection by dye conjugated streptavidin Proteins used in the assay are not denatured and their native tertiary structures are intact This may increase the chance for false negatives due to inaccessible binding residues on the protein Rev 11 3 Page 4 Antibody Array User s Guide Full Moon BioSystems Inc EXPERIMENTAL CONSIDERATIONS All reagents and materials are intended for research use only Handle the slides by holding the area with barcode labels Do not touch the slide surface Use extra care Any variation in buffers operator pipetting technique washing technique and incubation time or temperature can alter the performance of the kit Only use reagents and materials recommended by this user s guide Do not substitute buffers or solutions from other sources Do not allow arrays dry out between blocking coupling and washing It can cause high background Wash the arrays extensively with Wash Solution and water to remove excess residual reagents from the slide surface The reagents provided in the Antibody Array Assay Kit do not contain protease inhibitors To prevent protein degradation you should work quickly and proceed diligently towards the array analysis step once you start the extraction Alternatively you may use inhibitors if you prefer or plan to store the proteins for a week or longer Always wear gloves before handling any reagents COMPONENTS Antibody A
10. e slide Make sure the slide is completely submerged Cover the Coupling Chamber Incubate on an orbital shaker rotating at 35 rom for 1 2 hours at room temperature Transfer the slide to a 100x15 mm Petri dish containing 30 ml of 1X Wash Solution See Reagent Preparation Increase shaker s speed to 55 rpm continue for 10 minutes at room temperature Discard the wash solution Repeat the wash step twice Rinse the slide extensively with Milli Q grade water as follows 1 Place the slide in a 50 ml conical tube Fill the tube with 45 ml of water Close the cap Shake for 10 seconds Discard the water 2 Repeat ten times Important It is critical to rinse the slide extensively to completely remove Coupling Solution from the slide surface After the last rinse cycle the layer of water left on the slide surface should appear uniformly smooth across the entire surface If it looks spotty it means the surface is not clean Please repeat Step 7 Shake off excessive water on the slide surface and proceed to the next step immediately Note Do not allow the slide to dry out G Detection Rev 11 3 Add 60 ul of Cy3 streptavidin 0 5 mg ml to 60 ml of Detection Buffer Pour 30 ml of Cy3 streptavidin Solution into a 100x15 mm Petri dish Submerge the slide in the Cy3 streptavidin solution Incubate on an orbital shaker rotating at 35 rpm for 20 minutes at room temperature in the dark or covered with aluminum foil Transfe
11. ipReader GE Healthcare formerly Typhoon 9410 9210 8610 Microarray Slide Amersham Biosciences Holder Kit Genewave AmpliReader 4600 Microarray Reader InDevr Vidia Microarray Imaging System INNOPSYS InnoScan Microarray Scanner 700 900 Autoloader Molecular Devices GenePix Microarray Scanner formerly Axon Instruments All models PerkinsElmer ProScanArray HT Microarray Scanner formerly Packard Bioscience ProScanArray Microarray Scanner ScanArray GX Microarray Scanner ScanArray GX PLUS Microarray Scanner Tecan LS Reloaded Versatile Scanner PowerScanner Microarray scanner Vidar Systems Revolution 4200 Microarray Scanner Image Analysis Array images can be analyzed by any image quantification software including analysis software provided by scanner manufacturers third party software or open source programs such as ImageJ Rev 11 3 Page 16 Antibody Array User s Guide Full Moon BioSystems Inc A GAL GenePix Array List file for each array is provided to aid image analysis Please note GAL files may not be compatible with a number of systems named above If your image analysis does not work can be downloaded from our website GAL files describe the size and position of blocks the layout of feature indicators in them and the names and identifiers of the printed substances associated with each feature indicator automatically generate grids for image analysis Rev 11 3 Page 17
12. iquot 3 4 ul of serum b Add Labeling Buffer to the sample to bring the volume to 75 ul Add 3 ul of the Biotin DMF solution to the sample Incubate the mixture at room temperature for 1 2 hours with vortexing every 10 minutes Note Store the rest of the Biotin DMF solution at 20 C for future use d Add 35 ul of Stop Reagent Mix by vortexing then quickly spin down Incubate for 30 minutes at room temperature with mixing e Proceed immediately to the next step or store the biotinylated sample at 80 C for future use E Blocking Rev 11 3 Pre blocking preparation Remove the Antibody Array pack from refrigeration Before opening the package allow the slides to warm up to room temperature 30 to 60 minutes Then open the package and allow the slides to dry for 30 or more minutes Depending on the ambient temperature and humidity adjust warm up and drying time accordingly 1 Add 30 ml of Blocking Solution See Reagent Preparation in a 100 x 15 mm Petri dish Important Make sure the solution is at room temperature and that the dry milk is completely dissolved before use 2 Submerge one slide in the Blocking Solution The side with a barcode label must face up 3 Incubate on an orbital shaker rotating at 55 rom for 30 to 45 minutes at room temperature Rinse the slide extensively with Milli Q grade water as follows 1 Place the slide in a 50 ml conical tube Fill the tube with 45 ml of water Close the cap Sh
13. lp Re blank Print Screen Recording Measurement complete 3 4 2010 9 52 AM Show Report O Overlay control Clear graph each Sample z 340 nm normalization V On ee 18 90 a Fi Sample ID Sample 1 Af 331 2 Abs 0 046 A 28010mmpath 4 573 Q o 2 o 2 lt o This i 260 280 1 27 wi 1 89 a 210 220 230 240 250 260 270 280 290 300 310 320 330 340 mgiml 4 57 Wavelength nm 3 5 2 Xxx 0 30 48 16 absorbance spectrum of a clear cell lysate One peak is observed at 230nm and a second peak is observed at 270nm The two peaks are clearly separated This shows that the lysate is clear and ready for the next step Proceed immediately to the next step Protein Labeling or store the lysate at 80 C Page 12 Antibody Array User s Guide Full Moon BioSystems Inc D Protein Labeling Biotinylation of Protein Samples 1 Biotin Preparation a Briefly centrifuge Biotin Reagent before use b Add 100 ul of DMF N N Dimethylformamide to 1 mg of Biotin Reagent It will give a concentration of 10 ug ul Mix by vortexing then quickly spin it down Label this solution as Biotin DMF 2 Labeling a Aliquot 10 25 ul of lysate It should contain 80 150 OD of protein If BCA or Bradford assay was used to measure protein concentration the aliquot should contain 30 100 ug of protein When working with serum al
14. m any vendors including GE Healthcare PA43001 Invitrogen S 32355 Alexa Fluor 555 streptavidin Sigma Aldrich 6402 and others o Other dye labeled streptavidin can be used in place of Cy3 streptavidin as long as the dye is compatible with your array scanner e 50 mL conical tube with cap e Centrifuge with 1 5 mL microtube rotor e Milli Q Grade Water or dd H2O e Orbital shaker e Petri dishes 100 x 15mm 9 cm in diameter e Slide drying only one option is required o By compressed air compressed nitrogen clean air supplied by a central line or from a cylinder Do not use canned air duster or heat gun blower o By centrifugation centrifuge with 50 mL swinging bucket rotor e UV Spectrophotometer e Vortexer e Microarray scanner compatible with 3 x 1 inch 76 x 25 mm slides Optional o If you don t have access to a scanner you can send the finished array slides to our lab for scanning Please visit our website for details Rev 11 3 Page 7 Antibody Array User s Guide Full Moon BioSystems Inc REAGENT PREPARATION REAGENTS TO WARM BEFORE USE Blocking Reagent Coupling Reagent Warm to 25 30 C in a water bath Wash Buffer Biotin Detection Buffer DMF Dry Milk Labeling Buffer Stop Reagent Warm to room temperature 2 RXN Kit KASO2 i 1X Wash Make 1 10 dilution In a one liter reagent bottle add 100 ml of 10X Wash Solution Buffer to 900 ml of dd H20 Shake to mix 2 Blocking
15. nd Array Layout Multiple positive markers and negative controls are included in each array Positive markers contain Cy3 labeled antibodies to mark the boundaries of the array Negative controls contain BSA Empty spots contain nothing and their signal intensity can be used as background in data analysis Each set of antibody arrays contains two slides two identical arrays one slide can be used for a control sample and the other for a treated sample GAL files are provided for each array and can be downloaded from www fullmoonbio com support gal ANTIBODY ARRAY ASSAY KIT The Antibody Array Assay Kit is designed for easy and reliable processing of Full Moon BioSystems antibody arrays It provides the major reagents required to perform protein extraction labeling conjugation and detection The reagents are convenient easy to use and optimized to work with our antibody arrays Each kit provides sufficient reagents to perform assays on two slides Rev 11 3 Page 3 Antibody Array User s Guide Full Moon BioSystems Inc HOW IT WORKS Protein Extraction from cells Ww tissues or bodily fluids o4 o le Biotinylation of Proteins w Protein Conjugation to Antibody Array Detection by Dye Streptavidin The ELISA based antibody array platform involves four major steps 1 Protein extraction with non denaturing lysis buffer 2 Biotinalyte the protein samples 3 Couple the labeled samples to the antibodies on the
16. ns simultaneously on a single slide saving precious resources and reducing the number of variables that affect experimental outcome Our unique collection of antibody arrays includes phospho specific arrays for studying phosphorylation events comprehensive exploratory arrays for examining hundreds of proteins in a single experiment and pathway arrays for studying highly relevant proteins in specific research fields Suitable samples include cell lysates fresh froze FFPE tissue lysates serum culture supernatant and bodily fluids ANTIBODY ARRAYS The antibodies are covalently immobilized on a high quality glass surface coated with our proprietary 3 D polymer materials to ensure high binding efficiency and specificity All arrays are printed on standard size microscope slides and each slide contains one complete array The arrays utilize fluorescent detection and can be scanned on all microarray scanners that are compatible with 76 x 25 x 1 mm 3 inch x 1 inch x 1 mm slides To maximize data reliability each antibody on the array is printed with replicates Pathway arrays contain six replicates Explorer Antibody Array Phospho Explorer Array and Signaling Explorer Array contain two replicates for each antibody To see a list of the antibodies featured in a specific array and their reactivity information please visit our website www fullmoonbio com and select the array of your choice Go to the Documentation section to view Antibody List a
17. r the slide to a new 100x15 mm Petri dish containing 30 ml of 1X Wash Solution Increase shaker s speed to 55 rpm continue for 10 minutes at room temperature Discard the wash solution Repeat the wash step twice Rinse the slide extensively with Milli Q grade water as follows Place the slide in a 50 ml conical tube Fill the tube with 45 ml of water Close the cap Shake for 10 seconds Page 14 Antibody Array User s Guide Full Moon BioSystems Inc Rev 11 3 Discard the water Repeat ten times Important It is critical to rinse the slide extensively to completely remove Detection Solution from the slide surface After the last rinse cycle the layer of water left on the slide surface should appear uniformly smooth across the entire surface If it looks spotty it means the surface is not clean Please repeat Step 6 Hold the slide with your fingers shake off excess water from the slide Dry the slide with compressed nitrogen or air or by centrifugation Note The goal is to remove water from the slide as quickly as possible By compressed air Do not use compressed air in a can for example desktop air duster Compressed air or nitrogen from a cylinder tank or an outlet on the fume hood is adequate Make sure the pressure is less than 40 psi Point the air nozzle at a 30 angle one inch away from the slide surface Starting from one end of the slide push the water off of the surface Repeat for the back side of the slide
18. rrays Material Reagent Quantity Purpose Storage Condition Antibody Microarray 2 slides Microarray 4 C 6 months Gal File 1 Data Analysis Online download User s Guide 1 Instructions Online download Rev 11 3 Page 5 Antibody Array User s Guide Antibody Array Assay Kit sold separately from the arrays Full Moon BioSystems Inc Catalog No Description Quantity KAS02 Antibody Array Assay Kit 2 Reactions 2 Reactions KAS20 Antibody Array Assay Kit 20 Reactions 20 Reactions Material Reagent PAAD Purpose STorage 2 Rxn Kit 20 Rxn Kit Condition Biotin Reagent 1 mg 5 x 1mg Labeling 20 G Blocking Reagent 60 mL 600 mL Blocking 4 C Coupling Chamber 1 5 Coupling RT Coupling Reagent 12 mL 120 mL Coupling 20 G Detection Buffer 60 mL 600 mL Detection 4 C DMF 200 uL 1mL Labeling 4 C Dry Milk 1 8g amp 0 36g 18g amp 3 6g Coupling 4 C Extraction Buffer 1 5 mL 15 mL Cell and tissue lysis 4 C Labeling Buffer 2 mL 20 mL Labeling 4 Lysis Beads 2 tubes 20 tubes Cell and tissue lysis RT or 4 C Spin Columns 2 sets 20 sets Buffer Exchange RT Stop Reagent 100 uL 1 mL aera 4 C 10X Wash Buffer 100 mL 500 mL x 2 Washing 4 C Rev 11 3 Page 6 Antibody Array User s Guide Full Moon BioSystems Inc ADDITIONAL MATERIALS REQUIRED e 1X PBS pH 7 4 e Dye conjugated streptavidin o Cy3 Streptavidin is commonly used It can be purchased fro
19. should appear white and the PBS wash solution should appear clear and colorless Add one tube of Lysis Beads to 10 40 mg of tissues Add Extraction Buffer to the tissues The amount of Extraction Buffer needed should be determined by the amount of tissue harvested For 10 20 mg of tissue use 100 uL of Extraction Buffer for 20 40 mg of tissue use 200 uL of Extraction Buffer Vortex rigorously for 30 seconds to 1 minute Incubate on ice for 10 minutes Repeat five times Centrifuge the mixture at 10 000 x g for 5 minutes at 4 C Transfer all liquid to a clean tube Discard the beads Centrifuge the new tube with liquid at 18 000 x g for 15 20 minutes at 4 C The supernatant on top should look colorless and transparent as water Transfer the clear supernatant to a clean tube Important If the supernatant appears to be cloudy centrifuge again at 18 000 x g for 15 to 20 minutes at 4 C Check again If the supernatant is still not clear store the lysate at 70 C for 10 to 20 minutes Immediately centrifuge at 18 000 x g for 15 to 20 minutes at 4 after removal from freezer Save the top clear layer and discard the rest The supernatant should look colorless and transparent as water Proceed immediately to Step B Buffer Exchange Lysate Purification Serum Plasma 1 2 Centrifuge the serum plasma sample at 18 000 x g for 10 15 minutes at 4 C Transfer 2 3 uL of the clear pale yellow liquid to a new tube and
20. uffers can be used However high concentrations of certain compounds adversely affect biotinylation of protein samples Be sure the lysis buffer contains no more than 50mM Tris 0 1 SDS 1 Triton X 100 or 1 NP 40 Please feel free to contact us if you are unsure about your lysis buffer Note The reagents provided in the Antibody Array Assay Kit do not contain protease or phosphotase inhibitors Commonly used protease and or phosphatase inhibitors such as Roche s inhibitor cocktail may be added to Extraction Buffer to prevent protein degradation or dephosphorylation l Cells 1 Adherent Cells Remove media and wash the culture with ice cold 1X PBS 3 5 times Remove remaining PBS and add 100 200 uL of Lysis Buffer to 1 to 5 million cells Detach cells using a scraper and transfer the cells and remaining supernatant to a clean microcentrifuge tube Add one tube of Lysis Beads Suspension Cells Transfer media containing cells to a clean tube Pellet the cells by centrifugation at 500 x g for 2 minutes at 4 C Remove media completely without disrupting the cells Wash the pellet with ice cold 1X PBS followed by centrifugation Repeat three times to ensure complete removal of media Discard supernatant Add Extraction Buffer and one tube of Lysis Beads to the cells The amount of Extraction Buffer needed should be determined by the number of cells harvested For 1 5 million cells use 100 200 uL of Extraction Buffer Important
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