2010 Products Catalog of Genomine,Inc.
Contents
1. Denatured Protein Native Protein A e Lysis buffer 10ml x 1 f e e Dilution buffer 20ml x 1 e Solution A 3ml x 1 e Solution B 5ml x 1 e e Dissolving Solution 8ml x 1 o _ e Delipidation Solution 10ml x 1 Procedure Summary 1 Native amp denatured Protein extraction Rey 2 Phosphoprotein specific complex forming v 3 Precipitation of phosphoprotein complex An 4 Dedelipidation and recovering of phosphoprotein Solution Pa P A ke Se a V Delipidation A NJ Protein precipitation 2 DE 1 D SDS PAGE analysis E analysis d s a gt 93 50ng Ordering information PhosPro kit 1 box 5 Reaction P5012 5 PhosPro kit 1 box 10 Reaction P5010 10 Fig 2 DE analysis of enriched phosphoprotein fraction from total denatured A C amp native protein B D stained with CBB A B and stained with ProQ Diamond staining C D CENO M INE Venture Bldg 307 Pohang TechnoPark 601 Jigok Pohang Gyeongbuk 790 834 KOREA Am TEL 82 54 223 2464 FAX 82 54 223 2460 E mail amkim genomine com PhosPep Phosphopeptide enrichment kit Isolation and enrichment of phosphopeptide from complex mixtures of enzyme digest of phosphorylated proteins Highlights of PhosPep kit Kit contents 50 reactions e Solution A e Solution B e Washing solution 4Xammonium acetate buffer e Dissolving solution 1 phosphoric acid e Phosphopeptide standard beta casein tryptic2. SOLUTION B 5ml x 1 DISSOLVING SOLUTION 8ml x 1 DELIPIDATION SOLUTION 10ml x 1 Additional Materials Required Methanol Ultrapure water Detecting Phosphorylated Proteins This phosphoprotein enrichment kit was optimized for the protein solution in denatured condition for example the samples prepared for 2 DE and can be applied to native proteins Enriched phosphorylated proteins could be detected by staining commercially available staining method using fluorescent dye or by probing with antibodies specific for phosphorylated proteins Procedure Summary 1 Protein extraction 2 Phosphoprotein specific complex forming 3 Precipitation of phosphoprotein complex 4 Dedelipidation and recovering of phosphoprotein Procedure for phosphoprotein enrichment from cell lysate Denatured protein condition 1 Add 300 600uI LYSIS SOLUTION to the cells or tissue and disrupt the cells and tissue by sonication or motor driven homogenation Adjust the volume of LYSIS SOLUTION in order the final concentration of extracted protein to be above 4mg m Vortex the cell lysate for 15min and centrifuge for 20min at 12 000 x g and save the supernatant Assay the protein concentration and dilute 2mg protein with DILUTION SOLUTION to be the final volume of 3ml Use 5ml tube Alternatively protein solution prepared for two dimensional gel electrophoresis could be directly used for enrichment by appropriately diluted wi
3. apoptosis and immune disorders Despite the importance and widespread occurrence of this modification profiling of phosphoproteins in cells is still a challenge due to the low copy of phosphorylated proteins in cell and the relative amount of phosphoproteins compared to unphosphorylated proteins Radiolabeling by ZP labeling is frequently used conventional method for investigation of phosphoprotein profile in conjunction with 2 DE or 1 D gel electrophoresis and autoradiogram Alternatively western blot analysis probed by phosphoprotein specific antibody is also used for this purpose Mass spectrometry has been shown to be a reliable and routine tool to identify proteins ina high throughput manner However the identification of phosphorylation by mass spectrometry is not a trivial matter and to this day is not routine also due to the low copy of phosphorylated proteins in cells Tel 82 54 223 2463 Fax 82 54 223 2460 Email dskimi genomine com http www genomine com Venture Bldg 306 Pohang Techno Park Pohang Kyungbuk Korea ROK This phosphoprotein enrichment and exclusion of unphosphorylated proteins provides advanced chance in detecting protein phosphorylation in gels with non radiolabeling method eg Staining with fluorescence dye and enables quantitative comparison between cells Kit contents 10 reactions LYSIS BUFFER 10ml x 1 DILUTION BUFFER 30ml x 1 Native Homogenation Buffer 30ml x 1 SOLUTION A 3ml x 1
4. 7 dk HPO HPO Hun 8 Ti A Fig 3 Identification of phosphopeptide enriched from beta casein trypsin digest by PSD post source decay using MALDI TOF Panel A represents the MALDI TOF spectrum of enriched phosphopeptide from beta casein trypsin digest Blue asterisk represent the enriched phosphopeptides Rest of the seven spectrums represent the PSD spectrum of enriched phosphopeptide 16 Specificity and sensitivity of phosphopeptide enrichment Heterogeniety of phosphorylation site in peptide sequence could affect the efficiency of phosphopeptide enrichment We examined the specificity of Phos Pep kit for phosphopeptide from known phosphoprotein as a model peptide such as alpha casein ovalbumin pepsin and phosvitin As shown in Fig 4 Phos Pep isolated the phosphopeptide from most of the tryptic Trypsin digest s casen ovalbumin pepsin phosvitin A D j Lit Enriched phosphopeptide digest of model protein used and characterized as a phgosphopeptide by PSD using MALDI TOF The sensitivity of capability of Phos Pep was also tested From one microgram trypsin digest of beta casein which is equivalent to 45 pmole of each digested peptide to 175 fmole of 256 fold diluent the capacity of enrichment was tested Fig 5 FPS spectrum 1952 HPO HPO 2409 Fig 4 Phosphopeptide enrichment from trypsin digest of phosphoprotein E j 45pmole
5. 2 D Image yl Ee CD lege Fig 7 2 DE gel analysis of total extract of lung cancer cell line A enriched phosphoprotein pool from the extract of human lung cancer cell line B and enriched phosphoprotein pool from the extract of human lung cancer tissues c This result was represented in human lung cancer tissues which is composed more complex composition of cell types and body fluids As a result from the evaluation with phosphoprotein standards a casein B casein pepsin ovalbumin and phosvitin PhosPro technology showed phosphoprotein specific sensitive and high yield effective fractionation capabilities Table 1 Table 1 Phosphoprotein fractionation capability of PhosPro Evaluation category Specification Specificity 100 Selectivity gt 87 Sensitivity gt 93 50ng Yield gt 93 References 1 Alein L et al Proteomics 6 2157 2173 2006 2 Akira Yamagata et al Proteomics 2 1267 1276 2002 12 BS GENOMINE Por WITT through Genomic INSTRUCTIONS Phos Pep Phospho peptide enrichment kit Product Number P 5010 Storage Temperature 10 to 37 C RT INTRODUCTION Protein phosphorylation is one of the most frequently occurred posttranslational modification and plays a critical role in cellular regulatory events Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine threonine and tyrosine residues Despite the importance and w
6. 2010 Products Catalog of Genomine lnc Thy best research partner in proteomics THY BEST RESEARCH PARTNER IN PROTEOMICS i J GENO M INE Venture Bldg 307 Pohang TechnoPark 601 Jigok Pohang Gyeongbuk 790 834 KOREA a TEL 62 54 2723 7464 FAX 62 54 223 2460 E mail gmkim genomine com all Be WW Wwi http www genoproteom com 1 Overview PhosPro Phosphoprotein enrichment kit s0 1 PhosPep Phosphopeptide enrichment kit 2 Antibody biotin conjugation kit se 3 Peroxichrom TMB substrate solution 4 2 User manual amp Technical bulletin PhosPro User manual c cssccseseeceeceeeceeseeeeeeneeeeess 5 PhosPro Technical bulletin ccesesssssesessesseseeseeees 8 PhosPep User manual cscceccseseecseeeeeceeeeeeeeeeeeees 13 PhosPep Technical bulletin 15 Antibody biotin conjugation kit sass 19 Peroxichrom TMB substrate solution 21 ah C EN OM IN E Venture Bldg 307 Pohang TechnoPark 601 Jigok Pohang Gyeongbuk 790 834 KOREA TEL 62 54 223 2464 FAX 82 54 223 2460 E mail gmkimn genomine com SN http www genoproteom com Phosphoprotein enrichment kit PhosPro Highly selective and enrichment of phosphoprotein from complex mixtures of cell lysate Highlights of PhosPro kit Kit contents 10 reactions
7. A Cat D5015 100 Peroxichrom 100ml D5015 400 Peroxichrom 400ml D5016 100 peroxichrom Excel 100ml D5016 400 peroxichrom Excel 400m Highlights of TMB substrate 8 Sensitivities comparison of Peroxichrom Excel gt Ready to use single component gt Highest sensitivity 4 000 gt Sufficient dynamic range 3 500 3 000 gt Easy to use i amp 2500 2Cstandard TMB 1 gt Noncarcinogenic z E 2C st andard TMB 2 2 000 kk 1C PeroxiChrom3 gt No DMF or DMSO present in the reagent Ee EEN gt Stable at RT 1 000 z 1C Peroxichrom Excel6 0 500 gt Easy to transport 0 00 E 00 100 05 2 00 E 05 3 00 05 4 00 05 5 00 E 05 6 00 05 Peroxidase serial dilution from 1 50 000 of Img ml streptavidin HRP Description TMB substrate 3 3 5 5 tetramethylbenzidine is a chromogen that yields a deep blue color maximum absorbance at 605nm or 650nm when oxidized with hydrogen peroxide catalyzed by HRP The color then changes to yellow with the addition of 2N HSO with maximum absorbance M e at 450 nm Our TMB Substrates Peroxichrom Peroxichrom Excel are one component M and substrates that require no preparation before using stable and sensitivity Also Peroxichrom Peroxichrom Excel contain no organic solvents such as DMF DMSO methanol so there is no issue of safety with user Precautions TMB substrate is sensitive to contamination from a variety o
8. E HOMOGENATION BUFFER SOLUTION A SOLUTION B DISSOLVING SOLUTION DELIPIDATION SOLUTION Procedure Summary 1 Native amp Denatured Protein extraction 2 Phosphoprotein specific complex forming 3 Precipitation of phosphoprotein complex 4 Delipidation and recovering of phosphoprotein Denatured Protein E e ey Total cellular proteins p or P Solution A A 1 f p Solution B We Solution D D Delipidation amp A Protein precipitation JS Zeg D 2 DE 1 D SDS PAGE analysis D p Native Protein Additional Materials Required _ Methanol _ Ultrapure water Detection of Phosphorylated Proteins This phosphoprotein enrichment kit was optimized for the protein solution in denatured condition for example the samples prepared for 2 DE and can be adapted to native proteins Enriched phosphorylated proteins could be detected by staining commercially available staining method using fluorescent dye or by probing with antibodies specific for phosphorylated proteins Results and Discussion Phosphoprotein enrichment from Saccharomyces cerevisiae protein extract A CBB staining 123456 8 9101112191415 ji i i B ProQ Diamond staining Hi eseo Batimme AE ob Fig 2 SDS PAGE analysis of enriched phosphorylated proteins by PhosPro A Coomassie Brillant Blue SIGMA staing B ProQ Diamond Invitrogen staining Lane 1 7 The supernatant containing unphosphorylated prote
9. ER 11 pmole Ka WM D Al Alay i 2 8 pmole K Fins asd il i 730 fmole HK Waas di 175 fmoale Fig 5 Sensitivity of enrichment of phosphopeptides Phosphorylation site determination from enriched phosphopeptide Beta casein 2062 phosphopeptide CAF sequencing FQpSEEQQQTE Fig 6 Determination of phosphorylation site of beta casein phosphopeptide enriched using Phos Pep followed by chemical assisted fragmentation CAF Phosphopeptide enrichment from beta casein trypsin digest mixed with pTyr synthetic phosphopeptide x 1055 x 5K 3 oF i we A air Lah Jin V Wes Pitti let hel FA t When ob Aa we ein d PSD spectrum of 1055 m z pTyr Asp Leu Leu Leu Glu Fig 7 Enrichment and phosphorylation site determination of phosphopeptide containing phosphotyrosin amino acid residue 18 E Tel 82 54 223 2463 Fax 82 54 223 2460 s E JM N e Email alloyo02 genomine com WEI http www genomine com Venture Bldg 306 Pohang Techno Park Pohang Kyungbuk Korea ROK INSTRUCTIONS Antibody Biotinylation Kit Additional Materials Required Product Number P 5013 Ultrapure water Store at RT 80 Glycerol Optional Alternatives For Reactive Biotin ES BEZE Reactive Biotin NHS Biotin could be replaced Biotin is used in two step detection systems in by commercially available activated biotin concert with conjugated avidin Biotin is typically c
10. TOF mass spectrum of beta casein trypsin digest upper and enriched phosphopeptide from beta casein trypsin digest using Phos Pep lower Blue asterisk represent the enriched phosphopeptides Phosphopeptide identification Beta casein was used to examine the phosphopeptide is a major component of capacity of Phos Pep non IMAC commercially available alpha casein phosphopeptide enrichment kit Beta casein phosphopeptide see Fig 4 Some peptides purchased from Sigma Cat No C6905 was ranging 2884 to 3054 seems to be the digested with trypsin and phosphopeptide derivatives of tetra phophopeptide of 3124 was enriched using Phos Pep and peptide which have differential mass according to the map was measured Phosphopeptide which status of the number of phosphorylation site was not shown in total digest of beta Some of phosphopeptide was identified by casein Fig 2 was detected in enriched PDS of MALDI TOF by detecting the mass fraction Fig 2 lower panel Fig 3 A Among loss of phosphoric or phosphorous acid these phosphopeptide mono Fig 3 phosphopeptide 2062 and tetra phosphopeptide 3124 was originated from beta casein Some phosphopeptide 1660 and 1952 seems to be originated from contaminated alpha casein because this Intensity i 7 aia HD 2025 S 2054 HPO 1952 Pat HPO 3 H4 wem H4PO s Au Tt HePO ge e al a i EE ER a HPO HPO 2925 2124 KZ 3 ong git H PO
11. al bulletin http www genomine com Kit contents 50 reaction Solution A Solution B Washing solution 4 lt ammonium acetate buffer Dissolving solution 1 phosphoric acid Phosphopeptide standard beta casein tryptic digest 10ug Additional Materials Required C18 microtip Ultrapure water Binding Capacity In binding assays performed using this product binding of greater than 90 pmoles of phosphopeptide per 10ul of A solution is observed in one reaction From 1119 45 pmole trypsin digest of b casein single enrichment recover over 95 mono 2062 Da and tetra 3124 Da phosphopeptide Procedure Summary 1 Selective binding of phosphpeptide 2 Precipitation of phosphopeptide complex 3 Washing phosphopeptide aggregates 4 Dissolving of phosphopeptide aggregates Procedure for phosphopeptide enrichment from trypsin digest 1 Prepare trypsin digest 2 Add 5ul of solution A to 10ul trypsin digest in microcentrifuge tube and vortex briefly for a few seconds then stand about 1 5min 3 Add Gul of solution B to the previous mixture and vortex well to disperse the aggregate to homogeneous cloudy suspension Stand at least 30min until the cloudy aggregate settle down and disappear When cloudy aggregate was not disappeared within 30 min stand prolonged time till the aggregate disappear completely 4 Discard solution to the last drop with pipet tip Phosphopeptide aggregates was stuck to the wall and re
12. containing EDTA Dialysis the enriched phosphoprotein solution with appropriate buffer solution Or skip to the next step for preparation of 1D or 2D electrophoresis samples 5 Add 750 ul of delipidation soln methanol chloroform 600 150 and vortex vigorously for 5 min and centrifuge at 12 000rpm for 10 min for phase separation of solution Recover the middle phase protein disk and discard lower and upper phase solution completely Then wash the protein disk with sufficient 1ml methanol for two times 6 Dry the protein pellet in air or oven completely and dissolve the protein pellet with the solution for 2 DE electrophoresis or 1 D SDS PAGE Optimization of Results When you start with cell lysate at higher concentration and smaller volume of proteins use SOLUTION A and SOLUTION B 80uI and 120uI per 1ml protein solution respectively References 1 Philip Cohen Eur J Biochem 568 2001 2010 2001 2 Alein L et al Proteomics 6 2157 2173 2006 Related Products Product Code Phospep Phosphopeptide enrichment kit Technical bulletin Tel 82 54 223 2463 Fax 82 54 223 2460 http www genomine com venture Bldg 306 Pohang techno park Pohang kyungbuk Korea ROK ott GENO e NE senomis Enrichment and identification of phosphopeptide using PhosPro Introduction PhosPro phosphoprotein enrichment kit was developed to fractionate phosphorylated proteins from protein mixtures s
13. des Investigators to characterize the phosphoproteins by mass spectrometry hampered by the low abundance of phosphoprotein and the suppression of ionization of phosphopeptide in mass spectrometry resulting in failure to obtain sufficient signals Phos Pep facilitates the isolation and enrichment of phosphopeptide from complex mixtures of trypsin digest of phosphorylated proteins Strategy Phosphopeptide Enrichment Phos Pep kit De novo Sequencing Phosphopeptide Phosphoprotein Phosphorylation Site Identification Enrichment Determination PepFrag kit PSD CID PhosPro kit Fig 1 Phosphopeptide enrichment using Phos Pep in conjunction with MALDI TOF based CAF sequencing or MS MS for identification of phosphopeptide and phosphorylation site determination Materials amp Methods Materials Phos pep kit contents Solution A Solution B Washing solution ammonium acetate stock solution Dissolving solution 1 phosphoric acid Phosphopeptide standard beta casein tryptic digest 10g Procedure Summary 1 Selective binding of phosphpeptide 2 Precipitation of phosphopeptide complex 3 Washing phosphopeptide aggregates 4 Dissolving of phosphopeptide aggregates 5 Desalting and concentration 6 Characterization of phosphopeptide Results and Discussion Phosphopeptide enrichment from beta casein Beta casein total lysate Phos pep Non IMAC enrichment a rer EA ee ai al I i Fig 2 MALDI
14. digest Procedure Summary 1 Selective binding of phosphpeptide Fig Identification of phosphopeptide enriched from beta casein ek trypsin digest by PSD post source decay using MALDI TOF 2 Precipitation of phosphopeptide complex Panel A represents the MALDI TOF spectrum of enriched Washin f oh h ti r t phosphopeptide from beta casein trypsin digest 3 ok le EE de EE Blue asterisk represent the enriched phosphopeptides 4 Dissolving of phosphopeptide aggregates Rest of the seven spectrums represent the PSD spectrum of enriched phosphopeptide Solution A Solution B e Dissolving solution d Mass spectrum _ Ordering information PhosPep kit 1 box 50 Reaction P5010 GE NO MINE Venture Bldg 307 Pohang TechnoPark 601 Jigok Pohang Gyeongbuk 790 834 KOREA a TEL 82 54 223 2d64 FAX 82 54 223 2460 E mail gmkim genomine com Ao E http www genoproteom com Antibody biotin conjugation kit One stop labeling of Antibody Purification to Biotinylation Highlights of Biotinylation kit Ka Only 1 hour to get conjugates No filtration tube required amp High recovery amp consistent Applicable for 20ug to 10mg IgG Kit contents 10 reactions j l e Antibody purification acetate Gan 1M Sodium Acetate DAD tml x 1 Carprylic Acid Oom x 1 Neutralizing Buffer Iml x 1 e Desalting amp buffer exchange Solution A 8ml x 1 Labeling Bu
15. e phosphate moiety on the proteins by examining the changes after treatment of phosphatase The PPase was used as a phosphatase as previously described Prod staining CBB staining Fig 6 Phosphoprotein staining of enriched phosphoproteins and dephosphorylated proteins Lane standard phosphoproteins lane2 standard phosphoprotein treated with APPase lane3 supernatant fraction remained from phosphoprotein enrichment lane4 phosphoprotein fraction enriched by PhosPro lane5 phosphoprotein fraction treated with APPase As shown in Fig 5 the proteins in enriched phosphoprotein fraction was stained with phosphoprotein staining whereas the same proteins treated with phosphatase APPase was not stained with phosphoprotein staining but only with CBB staining This result represent that the proteins stained with phosphoprotein staining was phosphate group specific and the PhosPro isolate the phosphoproteins from cell extract in a specific manner Enrichment of low abundant phosphoproteins from lung cancer cell 11 lines and lung cancer tissues In 2 DE gel analysed using 300ug proteins of total cell lysate of lung cancer cell lines h460 about 35 protein spots was stained with phosphoprotein staining When the phosphoproteins 300ug were enriched from total protein extract of lung cancer cell line 198 protein spots were detected as a phosphoproteins Fig 6 B CBB staining Prod staining On el Rae
16. f oxidizing agents Avoid prolonged exposure to light contact with metal or air we recommend using TMB substrate by pouring out required amount into a reservoir and do not return excess TMB to provided bottle Storage Stable at 4C or Room temperature for 1 year Procedure 1 Warm to room temperature prior to use if you store TMB solution at A 2 Dispense 1 Component appropriate TMB solution or 100ul into each well 3 After sufficient color development 5 10minutes at room temperature or at 37C add 50ul Stop Solution 2N H2SO 4 or 1M H3PO4 to each well 4 Read plates at 450nm 21
17. ffer 6ml x 1 e Biotin labeling Reactive Biotin x 1 Stop Solution O 3ml x 1 gi Desalting and Buffer Exchange B Add Solution A and precipitate the immunoglobulin aggregate Dissolve the precipitated Procedure Summary immuneglobulin with Labeling buller Biotin Labeling sechert 1 Antibody Purification Reactive Biotin ez 9 2 Desalting and Buffer Exchange vs ba a 3 Biotin labeling gf Add Stoo solution A Stopping and Storage D Ordering information Antibody biotin conjugation kit 1 box 10 Reaction P5014 GENO A INE Venture Bldg 307 Pohang TechnoPark 601 Jigok Pohang Gyeongbuk 790 834 KOREA ae TEL 82 54 223 2d64 FAX 62 54 223 2460 E mail amkim genomine com es 3 http www genoproteom com Peroxichrome H Peroxichrome Excel TMB Peroxidase substrate solution 1 Component for ELISA Highlights of TMB substrate kit e Ready to use single component e Highest sensitivity Sufficient dynamic range Easy to use Noncarcinogenic No hydrogen peroxide required No DMF or DMSO present in the reagent Stable at RT Ease of shipping Pricing TMB substrate 3 3 5 5 tetramethylbenzidine is a chromogen that yields a deep blue color when oxidized with hydrogen peroxide catalyzed by HRP The color then changes to yellow with the addition of 2N H2S04 with maximum absorbance at 450 nm Our TMB Substrates Peroxichrom peroxichrom Excel are one component subs
18. idespread occurrence of this modification identification of protein phosphorylation site is still a challenge due to the low copy of phosphorylated proteins in cell even when performed on highly purified protein Mass spectrometry has been shown to be a reliable and routine tool to identify proteins ina high throughput manner However the identification of phosphorylation sites by mass spectrometry is not a trivial matter and to this day is not routine The detection of phosphopeptides by mass spectrometry in a complex mixture such as a tryptic mass fingerprint is a rare occurrence This is thought to be caused by suppression of the ionization of the mainly negatively charged phosphopeptide in the presence of a large excess of nonphosphorylated peptides This phosphopeptide enrichment kit provides highly selective enrichment of phosphopeptide from non phosphorylated complex trytic digest of proteins and facilitates the identification of Tel 82 54 223 2463 Fax 82 54 223 2460 Email alloyoO2 genomine com http www genomine com Venture Bldg 306 Pohang Techno Park Pohang Kyungbuk Korea ROK phosphopeptide by mass spectrometry Phosphopeptde identification relies on measuring the loss of mass Phosphopeptides tend to lose their phosphate group as phosphoric H3PO or phosphorous acid HPO3 due to metastable decay in MALDI TOF ESI PSD Jon trap CID or as phosphorous acid HPO by phosphatase Technic
19. ins discarded in step3 See Procedure Summary above Lane 8 15 enriched fraction containing phosphorylated proteins Independent seven trials for phosphoprotein enrichment were performed and analysed by SDS PAGE In order to evaluate the performance of PhosPro yeast protein extract was used The resulting enriched phosphorylated protein was analysed with 1 D Fig 2 or two dimensional gel electrophoresis 2 DE Fig 3 A total protein CBB staining e B enriched protein CBB staining C enriched protein ProQ staining Fig 3 2 DE analysis of total protein A enriched phosphoprotein fraction stained with CBB B and stained with ProQ Diamond staining C As shown in Fig 2 and Fig 3 most of the proteins stained with ProQ Diamond was detected in enriched phosphoprotein fraction This result represent that the PhosPro is highly specific for phosphoprotein fractionation Phosphoprotein enrichment from mouse brain native protein extract Enriched Native protein Enriched Denatured protein Fig 4 2 DE analysis of enriched mouse brain phosphoprotein fraction from total denatured A C amp native protein B D stained with CBB A B and stained with ProQ Diamond staining C D In order to evaluate the performance of PhosPro mouse brain denatured A C and native protein B D extract was used as a starting material The resulting enriched native phosphorylated protein was analysed using 2 DE Fig 4 A
20. mained coated in microcentrifuge tube This aggregate can be stored for several days 5 Add 50u of 1X washing solution dilute stock solution four fold and vortex briefly for a few seconds then discard washing solution completely 6 Add 10ul dissolving solution and stand at least 5min You can see sometimes some bubble gas formed along with the surface of microcentrifuge tube 7 Vortex briefly for a few seconds and if necessary dissolve the remaining crystals with pipetting 8 Desalt or concentrate the solution with C18 microtip column for mass spectrum analysis Optimization of Results 14 When peptide solution contains high salt dilute the solution below 100mM of salt prior to enrichment to obtain better result References 1 Joerg R et al Proteomics 4 3686 3703 2004 2 Metzger S and Hoffmann R J Mass Spectrom 35 1165 1177 2000 3 Hoffmann R et al J Mass Spectrom 34 1195 1204 1999 4 Akira Yamagata et al Proteomics 2 1267 1276 2002 Related Products Product Code Phos pro Phosphoprotein enrichment kit P5012 Technical bulletin Tel 82 54 223 2463 Fax 82 54 223 2460 http Awww genomine com venture Bldg 306 Pohang techno park Pohang kyungbuk Korea ROK GENOMINE ad ife through Genomic E E WW Enrichment and identification of phosphopeptide using Phos Pep Introduction Phos pep H was designed to ensure the charactrization of phosphopepti
21. onjugated to proteins via primary amines i e lysines Usually between 3 and 6 biotin molecules are conjugated to each antibody Procedure Summary 1 Antibody Purification 2 Desalting and Buffer Exchange 3 Biotin labeling 4 Stopping and Storage The entire conjugation can be performed in about an hour In general you will need to have a solution of your antibody at a concentration optimally of at least 2 mg ml The extent of biotin conjugation to the antibody may depend on the concentration of antibody in solution for consistent conjugations use a consistent concentration This product provides convenient and efficient method for removing salt and amine compound interfering biotin coupling reaction from antibody solution Procedure Antibody Purification by Carprylic Acid precipitation 1 Add 500u of DW to 500uI serum and acidify by adding 60u of 1M sodium acetate pH 4 0 Kit contents 10 reactions 2 Slowly drop wise add 37 ul 20ul for rat or mouse serum of caprylic acid and continue 1M Sodium Acetate pH4 0 Imlx1 stirring for 20 min at room temperature Carprylic Acid 0 5ml x 1 Neutralizing Buffer imix 1 3 Centrifuge at 12 000xg for 10min and Solution A 8ml x 1 carefully remove and save the supernatant Labeling Buffer 8ml x 1 DMSO 0 41mi x1 4 Adjust the pH 7 5 8 5 of supernatant antibody solution by adding 50u of neutralizing Reactive Biotin 1 solution and if necessary centrifuge the Stop Solu
22. s shown in Fig 4 D most of the proteins stained with ProQ Diamond in denatured protein fraction was detected in enriched phosphoprotein fraction from total native protein But native phosphoprotein fraction contains more non phosphorylated proteins not stained with ProQ Diamond which is assumed as subunit or proteins interacting with phosphorylated proteins than denatured phosphoprotein fraction As a result PhosPro is applicable to both denatured rather pure phosphoprotein fraction and active phosphylated protein fractionation Phosphoprotein identification by mass spectrometry The protein spot enriched and stained with ProQ Diamond and identified as a phosphoprotein spot N 3405 in Fig 3 B and C was further confirmed by mass spectrometry Protein was identified by MALDI TOF based peptide mass fingerprinting Its phosphopeptide was enriched by PhosPep and identified with MALDI PSD by detecting the loss of phosphoprous group 98Da from mother phosphopeptide m z 2900 392 A j l AH A Ny di WW C Fig 5 Identification of enriched phosphoprotein by mass spectrometry Phosphoprotein identification by dephosphorylation using APPase The phosphoproteins were enriched by PhosPro from cell lysate of h460 lung cancer cell line The enriched protein fraction which was stained with phosphoprotein staining and presumed to be the phosphoproteins was confirmed whether the staining was derived from th
23. sphorylated protein from cell lysate in native conformation of proteins omit step 1 using LYSIS SOLUTION Instead prepare the cell lysate with Native Homogenation Buffer or appropriate buffer solution except solution including phosphate 1 Add 1 3ml Native Homogenation Buffer to the cells or tissue and disrupt the cells and tissue by sonication or motor driven homogenation in order the final concentration of extracted protein to be 2 30mg ml Centrifuge for 20min at 12 000 x g and save the supernatant Use1 or 5ml tube 2 Add 80uI of SOLUTION A per 1ml protein solution and mix gently for 15min by inverting or gentle vortexing After subsequent adding 120u1 SOLUTION B per 1ml original solution incubate for 15min by gentle inverting then centrifuge briefly for 5 10 min for the aggregated materials to be settled down And discard upper clear solution 3 Add one fourth volume of Native Homogenation Buffer to the pelletted phosphoprotein complex and wash the residual non phosphorylated protein solution by resuspension the pellet and recover the pellet by brief centrifugation Repeat this washing P5010 one time and save the aggregate in hard pellet 4 Add 0 7m DISSOLVING SOLUTION and dissolve the pellet by pipetting several times caution at this time COs gas will be formed Open lid and degas sufficiently and stand for 10 min the solution to be clear This solution contains enriched phosphoprotein in 250mM salt solution
24. th DILUTION SOLUTION 2 Add 240ul of SOLUTION A and rapidly mix by vortex vigorously for a few seconds then incubate for 15min by inverting or gentle vortexing After subsequent adding 3601 SOLUTION B and brief mixing incubate for 15min by gentle vortexing then stand still for 5 10 min for the aggregated materials to be settled down And discard about 4ml of upper clear solution 3 Transfer the remaining aggregate suspension to 1 5ml microcentrifuge tube and centrifuge the suspension at 12 000rpm for 5min Discard the supernatant and save the aggregate in hard pellet This aggregate can be stored for several days 4 Add 0 7ml DISSOLVING SOLUTION and dissolve the pellet by pipetting several times caution at this time COs gas will be formed Open lid and degas sufficiently before vortexing and vortex for 5 min 5 Add 750 ul of delipidation soln methanol chloroform 600 150 and vortex vigorously for 5 min and centrifuge at 12 000rpm for 10 min for phase separation of solution Recover the middle phase protein disk and discard lower and upper phase solution completely Then wash the protein disk with sufficient 1ml methanol for two times 6 Dry the protein pellet in air or oven completely and dissolve the protein pellet with the solution for 2 DE electrophoresis or 1 D SDS PAGE Alternative procedure for phosphoprotein enrichment from cell lysate Native protein condition If you want to isolate the pho
25. tion 0 3m X1 solution at 8 000xg for 5min and discard Solution B 8mlx1 precipitate 19 Alternatively start here if you have your own affinity purified IgG Desalting and Buffer Exchange 5 Add half volume of Solution A 0 5ml and mix thoroughly by gentle inverting then stand for 10 min 6 Precipitate the immunoglobulin aggregate by centrifugation at 12 000xg for 5min and remove completely the turbid supernatant 7 Dissolve the precipitated immunoglobulin with 300u Labeling Buffer to be approximate 2mg ml IgG 8 If there is any insoluble material discard it by high speed centrifugation for 10 min Biotin Labeling 9 Dissolve the Reactive Biotin with 60 ul DMSO 10mM in DMSO Aliquote and store at 700C Add Zul Reactive Biotin solution per 100ul IgG solution and incubate 30min at room temperature 10 At the end of the incubation add 2ul Stop Solution and subsequently add half volume of Solution B to aggregate the biotin labeled IgG and remove the residual reactive biotin Stand this suspension at 4 C for 20min 11 Centrifugation the suspension at 12 000xg for 10min and dissolve the aggregated IgG with equal volume of Labeling Buffer phosphate buffer Add 80 glycerol and store at 20 C 20 Tel 82 54 223 2464 E mail gmkim genomine com Venture Bldg 307 Pohang Techno Park 601 Pohang Gyeongbuk Korea Peroxichrom Peroxichrom Excel TMB peroxidase substrate solution 1 component for ELIS
26. trates that require no pre paration before using stable and sensitivity Also Peroxichrom peroxichrom Excel contain no solvents or organics such as DMF DMSO methanol so there is no issue of safety with user Sensitivities comparison of Peroxichrom Excel ELISA OC Ferechromn Emced 5 Perche Excel 6 3 TM B Su bst rate OD at 450nm 1 PFersachnom A B 1C Persxichmem 3 emmer SR 2C slardard TMB 2 SC siandard THE i Target Antigen RK 21 Component ggo O 00 E 00 1006 05 2006 05 3006 05 4006 05 500 05 6006 05 HRP linked Antibody Peroxidase serial dilution from 1 50 000 of Img ml streptavidin HRP Ordering information 1 box 100ml X 4 D5016 100 Venture Bldg 307 Pohang TechnoPark 601 Jigok Pohang Gyeongbuk 790 834 KOREA GENOMINE i NW 4 http www genoproteom com TEL 82 54 223 2d64 FAX 82 54 223 2460 E mail gmkim genomine com E A GENOMINE Fer WI ite through Genomile INSTRUCTIONS PhosPro Phosphoprotein enrichment kit Product Number P 5012 Store at RT INTRODUCTION Protein phosphorylation is one of the most frequently occurred posttranslational modifications and plays a critical role in cellular regulatory events Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine threonine and tyrosine residues In fact phosphorylation of proteins plays a key role in oncogenesis cell signaling
27. uch as cell lysate or body fluids and it is efficient to isolate and concentrate low copy phosphorylated proteins in cells This kit utilized proprietary phosphoprotein precipitation method instead using column or bead such as IMAC column or immobilized anti phophoprotein antibody beads Then in addition to its specificity for isolation of phosphoproteins it provides simple and convenient method for phosphoprotein fractionation and all processes are to be done with multi parallel samples in each one tube This kit was designed to use denaturant and detergent solution as the starting material of protein extraction in order phosphoprotein isolation not to be prevented by possible steric hindrance of phosphorylated moiety of proteins and not to be omitted by difficulty in solubilization of phosphoproteins embedded in membrane fraction or cell debris Strategy Phosphopeptide Enrichment Phos Pep kit a Phosphopeptide Identification PSD CID We Phosphoprotein Enrichment PhosPro kit De novo Sequencing Phosphorylation Site Determination PepFrag kit Fig 1 Phosphoprotein enrichment using PhosPro Protein phosphorylation could be identified by phosphoprotein specific enrichment in conjunction with phosphoprotein specific staining or MS based phosphopeptide identification and phosphorylation site determination Materials amp Methods Materials PhosPro kit contents LYSIS BUFFER DILUTION BUFFER NATIV
Download Pdf Manuals
Related Search