Improved spot resolution and detection of proteins in 2-D electrophoresis using 24 cm Immobiline DryStrip gels

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1 application note Immobiline DryStrip Improved spot resolution d detection of proteins in 2-D electrophoresis using 24 cm Immobiline DryStrip gels key words: Immobiline DryStrip 2-D electrophoresis ph gradient strip length sample load spot resolution In proteomic investigations, resolution d sample loading capacity are two importt factors for the successful comparison of complex protein samples by 2-D electrophoresis d especially for the detection of lowabundce proteins. Using 24 cm narrow-rge Immobiline DryStrip gels greatly improves resolution d sample loading capacity. This application note demonstrates the importce of strip length, ph gradient, d sample load of Immobiline DryStrip on resolution d number of detected spots in the second dimension of 2-D electrophoresis. Results show that the increased length d higher sample loading capacity of 24 cm IPG strips (ph ) give higher resolution compared with shorter IPG strips. Moreover, the high sample loading capacity of 24 cm IPG strips enables the detection of more spots compared with 7, 13, d 18 cm IPG strips spots Introduction Two-dimensional (2-D) electrophoresis is a key technology for the comparison of complex protein mixtures from biological samples in proteomics research. Immobilized ph gradient (IPG) strips with various ph rges d gel lengths offer high sample loading capacity, stable ph gradients in the first-dimension separation, d improved resolution d reproducibility. road ph gradient strips (3 1 or lab-cast 4 12 ph units [1]) are used to obtain overview of total protein distribution. Medium (4 7 d 6 9 ph units) d narrow (1 ph unit) ph gradient strips provide improved resolution d greater sample loading capacity. These narrow ph rge Immobiline DryStrip gels are particularly useful for detailed studies of proteins in the regions of interest since they c offer higher resolution (2) as well as greater loading capacity. Longer IPG strips (18 cm d 24 cm), especially those in the narrow ph rge, c also improve spot detection by offering improved resolution. The detection d alysis of 3397 spots Fig 1. 2-D alysis of mouse liver proteins on Ett DLT II 12.5 (26 2 cm) gels after narrow-rge focusing with () 18 cm d () 24 cm Immobiline DryStrip gels, ph 5 6. Sample loads were 1 µg protein for the 18 cm d 2 µg for the 24 cm IPG strip gels. pproximately 83% more spots were detected using 24 cm Immobiline DryStrip ph 5 6 th 18 cm Immobiline DryStrip. Gel images kindly provided by Prof. ngelika Görg, Technische Univerisität, Munich. low-abundce proteins c be further enhced by using 24 cm Immobiline DryStrip gels, covering a working ph rge from 3 to 1 (Fig 1). This study demonstrates the effect of IPG strip length, protein loading capacity, d ph gradient on spot detection d resolution in the second dimension of electrophoresis , p1

2 Products used mersham iosciences products used: Immobiline DryStrip ph 3 1, 24cm Immobiline DryStrip 24cm Immobiline DryStrip ph , 24cm Immobiline DryStrip 18cm Immobiline DryStrip ph , 18cm Immobiline DryStrip 13cm Immobiline DryStrip 7cm Multiphor II Electrophoresis Unit Ett Dalt II Separation Unit Hoefer SE 6 Stdard Vertical Unit PlusOne Silver Staining Kit, Protein PlusOne Coomassie lue PhastGel R-35 Tablets Methods 1. Investigation of effect of Immobiline DryStrip length, ph gradient, d sample load on b-lactoglobulin spot resolution Sample d 2-D gel electrophoresis ovine b-lactoglobulin (Sigma) was run on 13, 18, d 24 cm Immobiline DryStrip gels ph 4 7 d ph Sample load rged from 12.5 to 8 µg, depending on IPG strip length d ph gradient. Sample loads < 8 µg were cup loaded while larger sample amounts were applied during IPG strip rehydration. Immobiline DryStrip gels were run on Multiphor II Electrophoresis Unit. ll chemicals, reagents, d procedures used for 2-D electrophoresis are described in reference 4. Seconddimension SDS gel electrophoresis of Immobiline DryStrip gels was run on cm SDS gels on a Hoefer SE 6 vertical electrophoresis system. Vertical streaking on SDS gels was minimized by running gels at room temperature, initially at 25 m/gel for 3 min followed by 5 m/gel until the romophenol lue tracking dye reached the end of the gel. Staining Silver staining was performed according to instructions provided with PlusOne Silver Staining Kit, Protein. Staining was performed with Coomassie rillit lue according to reference 3. Gels were destained in a solution of 2% ethol d 5% acetic acid. Resolution Resolution Strip length 24 cm Strip length 18 cm µg β-lactoglobulin Strip length 13 cm Strip length 18 cm Strip length 24 cm 4 Fig 2. Spot resolution of b-lactoglobulin on SDS gels as a function of Immobiline DryStrip (ph ) length d sample amount µg β-lactoglobulin Fig 3. Spot resolution of b-lactoglobulin on SDS gels as a function of Immobiline DryStrip (ph 4 7) length d sample amount. Fig 4. SDS gel of b-lactoglobulin after IEF on 13 cm Immobiline DryStrip ph 4 7. (). 5 µg sample (). 1 µg sample. Fig 5. SDS gel of b-lactoglobulin after IEF on 18 cm Immobiline DryStrip ph 4 7. (). 1 µg sample (). 2 µg sample , p2

3 Fig 6. SDS gels of b-lactoglobulin after IEF on 24 cm Immobiline DryStrip ph 4 7. sample amount of 2 µg was loaded onto the IPG strip. Image alysis Evaluation of spots was performed using a 1-D scner d 1-D software package. Gels were scned using LabSc d evaluated using ImageMaster TotalLab 1D Gel nalysis, version 1. software. The software assigns peaks to each spot d the shape d height of each peak is dependent on spot size. Protein spot resolution between pairs of spots on the gel, R was determined according to the following equation: R = L [2/( + )] where, L = distce between two peaks = width of peak protein spot = width of peak protein spot The lowest value at which two spots could be considered well resolved was estimated to be 1.. This criterion was established by comparing visual data with resolution values for each spot pairing. Fig 7. SDS gels of b-lactoglobulin after IEF on 18 cm Immobiline DryStrip ph (). 4 µg sample (). 6 µg sample. Fig 8. SDS gels of b-lactoglobulin after IEF on 24 cm Immobiline DryStrip ph (). 6 µg sample (). 8 µg sample. 2. Investigation of the influence of Immobiline DryStrip length, ph gradient, d sample amount on number of protein spots detected (E. coli extract) Sample d 2-D electrophoresis Lyophilized E. coli cells (Sigma) were suspended in lysis buffer d lysed by sonication. The extract was run on 7, 13, 18, d 24 cm Immobiline DryStrip as well as, 18 cm ph d 24 cm IPG strips ph 3 1 d Sample loads < 8 µg were cup loaded while larger sample loads were applied during IPG strip rehydration. Immobiline DryStrip gels were run on Multiphor II Electrophoresis Unit. The second dimension was run on lab-cast 12.5% polyacrylamide gels containing a modified Laemmli buffer system. The 7 d 13 cm Immobiline DryStrip gels were run on cm SDS gels on a Hoefer SE 6 vertical electrophoresis system while 18 d 24 cm IPG strips were run on large-format SDS gels on Ett DLT II System. To allow direct comparison of SDS gels, run length in the second dimension was adapted to the length of the IPG strip, for example, 7 cm IPG strips were run 7 cm on the second dimension gel while 13 cm IPG strips were run 13 cm. Running conditions used are described in reference 3. Staining Silver staining was performed according to instructions provided with Plus One Silver Staining Kit, Protein. Coomassie staining was performed with Coomassie rillit lue according to reference 3, except for destaining in 7% acetic acid , p3

4 µl sample 2.5 µl sample 5 µl sample 8 µl sample 7 cm strip 13 cm strip 18 cm strip Fig 9. Number of spots detected by silver staining on SDS gels after IEF of E.coli on Immobiline DryStrip ph 4 7. Results 1. b-lactoglobulin spot resolution etter resolution was achieved with higher sample loads for the narrow-rge IPG strips (Fig 2) compared with mediumrge IPG strips (Fig 3). Resolution of protein spots decreased with increasing sample amount applied to both medium- d narrow-ph gradient Immobiline DryStrip gels of different length (Fig 2 d Fig 3). Resolution was improved with increasing length of Immobiline Drystrip (Fig 3). Fig 1. Number of spots detected by Coomassie staining on SDS gels after IEF of E.coli on Immobiline DryStrip ph µl sample 18 cm strip ph 3 1, 4 µl sample ph 3 1, 25 µl sample 35 µl sample 8 µl sample Fig 11. Detected silver-stained spots in ph interval on 24 cm Immobiline DryStrip ph 3 1, 4 7, d µl sample ph , 15 µl sample ph , 6 µl sample Fig 12. Detected Comassie-stained spots in ph interval on 24 cm Immobiline DryStrip ph 3 1, 4 7, d Image alysis of total 2-D map Gels were scned with LabSc. Scned gels were subsequently evaluated d number of spots estimated using ImageMaster 2D Elite software, version 3.1. Immobiline DryStrip 13, 18, d 24 cm Good resolution (R > 1.) on the SDS gel was achieved by applying up to 1 µg of b-lactoglobulin to 13 cm IPG strips (Fig 4). Increasing sample load to 2 µg resulted in poorer resolution with overlapping spots. Using 18 cm Immobiline DryStrip, good resolution was obtained up to 2 µg (Fig 5) d even better resolution was obtained for the 24 cm IPG strip (Fig 6). Immobiline DryStrip ph , 18, d 24 cm pplication of up to 6 µg sample to 18 cm Immobiline DryStrip, ph resulted in sufficiently high resolution (R > 1.) between spots (Fig 7). High resolution was achieved with up to 8 µg sample on 24 cm Immobiline DryStrip (Fig 8). 2. Number of spots detected versus gel length The number of spots detected from E. coli extract increased with increasing sample load d length of Immobiline DryStrip ph 4 7 on silver- d Coomassiestained gels (Fig 9 d Fig 1). The 24 cm IPG strip yielded 22% (silver-stained) d 18% (Coomassie-stained) more spots th the shorter 18 cm IPG strip. 3. Number of spots detected versus ph intervals Using narrower ph intervals d increasing the sample amount accordingly revealed more spots, both when using silver d Coomassie staining. In the ph interval (silver staining), 38% more spots were detected when chging from 24 cm Immobiline DryStrip ph 3 1 to Immobiline DryStrip ph 4 7 (Fig 11). The number of detected spots increased by a further 51% when chging from 24 cm Immobiline DryStrip ph 4 7 to Immobiline DryStrip ph For Coomassie-stained gels, a 3% increase in the number of detected spots was observed when chging from 24 cm Immobiline DryStrip ph 4 7 to ph (Fig 12) , p4

5 Conclusions Narrow-rge Immobiline DryStrip gels offer high sample loading capacity, as well as, high resolution d improved spot detection in the second dimension of 2-D electrophoresis. Using 24 cm narrow-rge Immobiline DryStrip gels in particular facilitates detection of low-abundce proteins. References 1. Görg,. et al., Electrophoresis 2, (2). 2. Lgen, H. d Röder, D. Life Science News 4, (2) D Electrophoresis using Immobilized ph Gradients, mersham iosciences, code number (1998) , p5

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