Agilent Protein sizing analysis using the Agilent 2100 Bioanalyzer Protein 200 LabChip kit
Contents
1. tion Some proteins such as bovine serum albumin give rela tively broad peaks compared to other proteins Resolution is also affected by the fact that some pro teins do not migrate according to their size Both effects are also observed using conventional SDS PAGE To confirm the theoretically calcu lated resolution of at least 10 a 10 gel protein mixture of eight different proteins some similar in molecu lar weight table 1 was analyzed as shown in figure 2B For exam ple carbonic anhydrase II 29 kD and I 81 kD which differ in size by only 6 5 were almost baseline separated using the Protein 200 assay However they were only partially separated using a 4 20 gradient gel figures 2 B and C 4 20 gel bioanalyzer ___ _ upper marker 210 kD Lied Daa iN phosphorylase b 97 4 kD up mf BSA 68 kD wee OValbumin 43 kD oan ee ee lactoglobulin cis one ee ysozyme 14 3 kD 18 4 20 1 17 31 17 18 4 U E 20 1 Figure 2 Resolution of the Protein 200 assay 45 18 4 kD 116 116 116 oor 116 45 31 z9 wee 17 18 4 20 1 e 14 A Analysis of the Protein 200 ladder with a linear gel a gradient gel and the Protein 200 assay B Electropherogram and the gel like image of a protein mixture of 8 different proteins Mw is indicated in kD demonstrating the resolution of the Protein 200 assay C Analysis of the same protei2. SDS PAGE based on 2 gels with n 20 as shown in figures 2B and C Protein Bioanalyzer Rel conc StDev CV ng pll lysozyme 117 4 12 7 10 8 myoglobin 9 4 2 3 248 3 lactoglobin 23 1 49 20 8 soybean trypsin inhibitor 22 5 3 8 17 0 carbonic anhydrase II 70 6 8 6 12 1 carbonic anhydrase 57 6 15 7 27 3 ovalbumin 34 6 2 2 6 3 3 galactosidase 26 2 1 5 59 Table 2 Reproducibility of the relative quantitation of eight different proteins with the Protein 200 assay and based on 5 chips with n 15 Conclusion The Agilent 2100 bioanalyzer is an ideal tool for quick and easy sizing and analysis of proteins For example the Protein 200 LabChip kit allows researchers to more efficiently monitor the purification process from cell lysates to the purified protein to study protein expression or to monitor antibody production purification and quali ty The use of internal and exter nal markers allows the analysis of multiple samples with excellent reproducibility and reliability Sep aration performance and data pre cision is comparable or superior to gel based analysis while analy sis times are greatly reduced Automation of separation and data analysis also makes the Agilent 2100 bioanalyzer versatile and easy to use In addition to the pro tein analysis the Agilent 2100 bio analyzer can be used in combina tion with a variety of other LabChip kits for the analysis of DNA and RNA samples Meike Kuschel is application che
3. Agilent 2100 bioanalyzer can be used with the Protein 200 LabChip kit to analyze a large variety of protein samples such as cell lysates column frac tions antibodies and purified pro teins Ten samples can be ana lyzed in less than 30 minutes This Application Note describes the performance of Agilent s Pro tein 200 LabChip kit in compari son to conventional SDS PAGE The Protein 200 LabChip kit offers several advantages over ls Agilent Technologies t Innovating the HP Way Experiment All proteins were purchased from Sigma Aldrich GmbH Taufkirchen Germany Dulbe co s phosphate buffered saline PBS was purchased from Life Technologies GmbH Karlsruhe Germany The Agilent 2100 bio analyzer Protein 200 LabChip kit and Protein 200 ladder and upper marker were obtained from Agi lent Technologies GmbH Wald bronn Germany All SDS PAGE reagents and gels were purchased from Invitrogen BV Groningen The Netherlands The digital camera and the imaging software were purchased from Kodak Digi tal Science Eastman Kodak Com pany Rochester NY USA Protein 200 assay The chip based separations were performed on the Agilent 2100 bioanalyzer in combination with the Protein 200 LabChip kit and the dedicated Protein 200 assay software All chips were prepared according to the protocol provid ed with the Protein 200 LabChip kit The kit includes 25 chips syringe spin filters and reagents In addit
4. Introduction With the Human Genome Project close to completion attention is now turning to the next step how to utilize this genetic data to better understand diseases and develop targeted therapeutics more efficiently Proteins rather than genes convey most cellular functions Understanding protein expression and protein function is crucial to the identification of new targets for drug development As we now enter this new post genomic era proteins will move into the focus of attention Although protein analysis tech nologies are developing fast many still rely on traditional methods such as SDS polyacrylamide gel electrophoresis SDS PAGE which are time consuming and Protein sizing and analysis using the Agilent 2100 Bioanalyzer and Protein 200 LabChip kit Application Meike Kuschel include a number of laborious manual steps With the increasing focus on proteins there is a strong demand to automate and speed up protein analysis The Agilent 2100 bioanalyzer uti lizing LabChip technology from SDS PAGE analysis These Caliper Technologies Corp is a include compact system for rapid and e significant time savings automated analysis of proteins e improved ease of use integrating multiple experimental e excellent reproducibility and procedures such as sample han e significant reduction of dling separation staining destain hazardous waste ing detection and analysis in a single process The
5. concentration of the pro teins within the sample are imme diately displayed in real time in the data table The same protein mixture shown in figure 2B was used to verify the sizing accuracy and reproducibili ty of the Protein 200 assay As shown in table 1 the sizing of the eight different proteins analyzed with the Agilent 2100 bioanalyzer was comparable to both the expected molecular weight and the molecular weight determined using SDS PAGE The sizing accu racy of SDS PAGE and chip based analysis depend on the protein characteristics and may therefore vary for particular proteins Some proteins may not migrate accord ing to their molecular weight In general the sizing reproducibility of the Protein 200 assay is excel lent commonly achieving a sizing reproducibility of 5 or better The concentrations of the proteins within this protein mix were determined with the Agilent 2100 bioanalyzer using a one point cali bration by comparing the peak area of the protein of interest to the peak area of the Protein 200 upper marker table 2 The quan titation reproducibility was below 30 The Protein 200 upper mark er is used for correction of differ ent injection efficiencies due to varying salt concentrations As indicated before the amount of material injected into the separa tion channel depends on the con ductivity of the sample buffer Because all proteins within a cer tain sample are affected to the same d
6. d an electropherogram for each sample figure 1 In contrast to conven tional SDS PAGE no additional manual staining destaining imag ing and analysis steps were neces sary Agilent Technologies 2100 Bioanalyzer Bio Sizing sm svs 2c_00032 File Edit View Graph Assay Tools Help A Ready internal markers Figure 1 oe lel amp E H z ne i Peak Number Data File sm svs 2 00032 Read 101000 2 23 07 Pri 7 Jaje b Sample 1 Sample 2 Sample 4 Sample 5 Assay Protein 200 neutral Sample 3 Sample 6 a LL Sample 7 Sample 6 Sample 10 Ladder Jaa dl Offline No serial port selected Sample 9 Auto Print Auto Export Agilent 2100 bioanalyzer software Data are displayed as gel like image as well as electropherograms samples 1 10 and Protein 200 ladder Pro tein samples with carbonic anhydrase 100 ng pl and bovine serum albumin in different concentrations 10 to 3000 ng l were analyzed The Pro tein 200 ladder first lane of the gel like image and the internal markers are used as reference for sizing and relative quantitation Sizing range and resolution Sizing range and resolution of the Protein 200 assay were evaluated and compared to the results obtained with conventional SDS PAGE analysis The Protein 200 ladder used for sizing with the Protein 200 LabChip kit was sepa rated and analyzed using both lin ear and gradient p
7. egree the use of an inter nal marker allows for correction of this variability and permits determination of the relative con centration of the proteins indepen dent of the sample matrix In con trast to batch based assays for quantitation based on methods developed by Lowry or Bradford the Protein 200 assay is compati ble with most of the commonly used protein sample buffers including a wide variety of reagents such as detergents dithiothreitol or imidazole Since absolute quantitation is only possible comparing individual results to a standard curve gener ated with the protein of interest the Protein 200 assay allows rela tive quantitation only However as observed with other protein dyes the relative quantita tion accuracy can vary from pro tein to protein due to the different staining efficiencies Protein Theoretical SDS PAGE Bioanalyzer Size kD Size kD StDev CV Size kD StDev CV lysozyme 14 0 14 0 0 5 3 3 13 8 0 4 2 9 myoglobin 17 0 17 6 0 5 3 0 16 2 0 6 3 7 3 lactoglobin 18 4 18 4 0 6 3 2 18 1 0 5 2 9 soybean trypsin inhibitor 20 1 19 1 0 6 3 4 20 7 1 0 5 0 carbonic anhydrase II 29 0 25 3 0 7 2 6 27 5 0 5 1 9 carbonic anhydrase 31 0 27 2 0 6 2 1 30 7 0 5 1 5 ovalbumin 45 0 46 1 1 1 2 3 42 4 0 5 1 2 3 galactosidase 116 0 116 6 7 8 6 7 118 3 0 6 0 5 Table 1 Sizing analysis of the same protein mixture including eight different proteins with the Protein 200 assay based on 5 chips with n 15 and
8. ion the Protein 200 ladder and upper marker were used The Agilent 2100 bioanalyzer is con trolled by intuitive and user friendly software which includes data collection reporting and interpretation functions SDS PAGE Gel electrophoresis was per formed with 10 and 4 20 Pre Cast Tris Glycine Gels according to the instructions provided by the manufacturer An equal volume Tris Glycine SDS sample buffer 2 x was added to the samples and they were denatured for 5 minutes at 95 C before loading onto the gel The separation was performed for approximately 100 minutes at constant 125 volts Gels were stained with a Colloidal Blue Stain kit and destained overnight A Kodak DC 1200 digital camera was used for imaging and analysis was performed with the Kodak 1D Image Analysis software Results and discussion Different protein samples were analyzed to verify the perfor mance of the Protein 200 assay with regard to size range resolu tion sensitivity linear dynamic range sizing and quantitation accuracy and reproducibility The Agilent s Protein 200 assay was used to determine the size and the relative concentration of each sample The results for each sam ple were viewed in real time as separation when detection was completed The first result was available in only seven minutes with each subsequent analysis fol lowing in 2 minute intervals The results were displayed in a tabular format a gel like image an
9. mist at Agilent Technologies Waldbronn Germany s LabChip is a US registered trade mark of Caliper Technologies J Corporation www agilent com chem labonachip Copyright 2000 Agilent Technologies All Rights Reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Published December 1 2000 Publication Number 5988 0975EN i Agilent Technologies Innovating the HP Way
10. n PBS as width of the separated protein shown in figure 3B For example band or peak this allows detection a 1 impuri ty close to a parent peak when The Protein 200 assay is linear checking the purification progress over two orders of magnitude for in column fractions example from 20 ng pl to 2000 ng pl BSA in PBS A correlation m A CA 1 BSA 10 ng pl BSA 2 50 ng pl CA 100 ng ul B Measured conc ng pll 3000 R 0 9679 i 2000 1000 0 1000 2000 3000 Actual conc ng ul Figure 3 Sensitivity and linear dynamic range of the Protein 200 assay A Gel like image and electropherogram showing the analysis of a protein sample with 100 ng jl carbonic anhydrase and 10 to 3000 ng pl BSA in PBS buffer B Graph showing the linear dynamic range of the Protein 200 assay Sizing and quantitation accu racy and reproducibility The Protein 200 ladder is run on each chip from a designated lad der well Following the analysis of the Protein 200 ladder the soft ware generates a calibration curve of the migration time versus the molecular weight of each protein contained in the ladder This cali bration curve is then used to determine the size of each of the detected proteins in the 10 sam ples The lower and upper mark ers which are run with each of the 10 samples correct for small drifts in migration time and ensure accurate sizing figure 1 Follow ing each analysis the size and the relative
11. n mixture with a 4 20 gradient gel scan and gel image Sensitivity and linear dynamic range To determine the sensitivity and the linear dynamic range of the Protein 200 assay protein samples containing 100 ng pl carbonic anhydrase and 10 to 3000 ng pl bovine serum albumin in phos phate buffered saline PBS were analyzed as shown in figure 3 The lower detection limit of the Pro tein 200 assay was 20 ng pl BSA in PBS 80 ng in 4 pl sample with an approximate signal to noise ratio of 3 Vendors state a sensitivity of 50 ng and 10 ng for standard Coomassie stain R 250 and col loidal Coomassie stain G 250 respectively Therefore the sensi tivity of the Protein 200 assay was comparable to the detection with a standard Coomassie stain How ever the sensitivity of the Protein 200 assay was effected by the salt concentration of the sample buffer If the sample buffer con tains salt concentrations lower than PBS a larger amount of pro tein will be injected into the sepa ration channel enhancing the sen sitivity Similarly increasing the salt concentration in the sample buffer will decrease the sensitivity of the Protein 200 assay for exam ple 40 ng pl of BSA in 0 5 M NaCl can be detected with a signal to noise ratio of 3 In addition the sensitivity of pro coefficient of R2 0 968 was deter tein detection is also affected by mined analyzing samples from 10 the staining efficiency and the to 3000 ng il BSA i
12. recast polyacry lamide gels and Agilent s Protein 200 assay figure 2A The Protein 200 assay was used to size and analyze proteins ranging in size from 14 200 kD under denaturing conditions in the presence of amp mercaptoethanol as a reducing agent The resolution of the Pro tein 200 assay was comparable or even better than the resolution that was achieved using a 4 20 polyacrylamide gel As expected the resolution of the linear gel was lower compared to the gradient gel It was also lower than the resolution achieved with the Pro tein 200 assay This was especially prominent in the low molecular weight range figure 2A Two pro teins lysozyme and b lactoglobu lin cannot be resolved on the 10 gel however they are clearly sep arated on both the gradient gel and Agilent s Protein 200 assay In addition the separation with the Agilent 2100 bioanalyzer resulted in sharper bands compared to the polyacrylamide gels figure 2A which allows more accurate siz ing Based on repeated measure ments with the Protein 200 ladder data not shown it was calculat ed that a resolution of at least 10 with a 50 valley can be achieved through the sizing range from 14 200 kD Above 100 kD resolution of 5 is achievable The resolution depends on the characteristics of the proteins within a sample and can be limit ed by several factors Protein het erogeneity that results in a larger peak width can reduce the resolu
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Agilent Protein sizing analysis using the Agilent 2100 Bioanalyzer Protein 200 LabChip kit