Zoom ipgrunner system manual

Spot quality is also an important parameter in 2DE analysis. We used ImageMaster 2D Platinum 7. This parameter is a measure based on the spot curvature. Real spots generally have high saliency values, whereas artifacts and background noise have small saliencies. The saliency is an efficient parameter for filtering and discarding spots, but it may also be used for the evaluation of the spot quality. For example, PDQuest software BioRad calculates spot quality numbers, which are mainly based on Gaussian fit assessing spot shape.

Absolute values of spot saliency may vary depending on brightness and contrast of 2D images. However, in our case all procedures of image processing for both IPG- and NEPHGE-based 2DE gels were the same, and therefore it was possible to compare two methods by using saliency as the spot quality parameter.

We have calculated the average saliency of protein spot and the percentage of low quality spots for every gel.

This data is provided in Tables 1 and 2. Average spot saliency for a whole gel at standard protein load was similar in both methods, but again there was a difference when comparing acidic and basic gel zones. To count low quality spots, we had set an arbitrary value of saliency to The saliency is highly dependent on the images, and, according to the software user manual, gels may need saliency values from 10 to for correct filtering.

The percentage of low-quality spots of acidic proteins at standard 1x protein load was considerably higher in NEPHGE-based method, whereas the results for spots of basic proteins were similar in both methods Table 1.

However, high protein load experiment showed substantially different results. Spot quality data confirmed that IPG strips were overloaded by 2x total protein amount. Thus, double protein load significantly decreased average spot saliency and increased percentage of low quality spots in IPG-based 2D gels, whereas NEPHGE-based 2D gels demonstrated increased overall spot quality see Table 2 and compare with Table 1.

Lower spot quality in standard protein load experiment may be at least partially explained by our imperfect performance with NEPHGE gels, which is reflected by higher error ranges of average saliency values than at high protein load Tables 1 and 2 , respectively. Anyway, high protein load onto NEPHGE gels is preferable, because both spot quality and reproducibility are excellent.

Further attempts to increase protein amount and detect even more spots in a small gel may result in overlaping of neighbouring proteins by spots of high-abundance proteins. We noticed that at least half of this variation is determined by different conditions in independent experiments. Thus, the easiest way to minimize the variation is to compare experimental sample with the control processed in parallel, but not in separate independent experiments. Experimental variation itself should not introduce false positive results when using larger amount of replicas, because in this case it is apparent in the error range.

However, it should be considered that the threshold of fold change for differentially expressed protein spot is at least 1. Lower fold changes would fall into experimental variation range and are unreliable values for differential expression. These thresholds should be considered at least when analysing whole cell lysates. Accordingly, the threshold for considering any protein spot as differentially expressed at high load should be the same as under standard conditions.

Both 2DE methods were examined in biological experiment on cytosolic unfolded protein response UPR-Cyto , and the results compared with our earlier study, where the same phenomenon was analysed using narrow range pH 4—7 Invitrogen IPG-based 2DE method [ 17 ].

The main differentially expressed protein spots, already identified and evaluated in previous experiment, are indicated by solid arrows in Figures 1 and 2 , and their quantitative analysis is given in Table 3.

We suggest that this may be partially related to different sample preparation and 2nd dimension electrophoresis in each method, because in our experiment overall protein pattern is rather similar, and most corresponding spots of high abundant proteins can be cross-referenced Figure 1. At standard protein load, both pH 3—10 methods were less sensitive than pH 4—7 method in the acidic pI range.

In this experiment, spots of Hspp were near the limit of detection, preventing reliable quantitative analysis, whereas Sgt2p and Sti1p were entirely undetectable using standard protein load in pH 3—10 based method. The increased expression of more abundant pI 4—7 proteins in UPR-Cyto was also determined by IPG pH 3—10 method; however, calculated fold changes were lower than in previous study Table 3.

New differentially expressed protein spots in the acidic pI range were not detected by either pI 3—10 range method. Lower sensitivity of pH 3—10 versus pH 4—7 IPG in the acidic zone could be compensated by the analysis in the basic pI protein zone. It can be noted that Sis1p was also identified as overexpressed cellular protein during UPR-Cyto in another earlier study using misfolded YFP expression [ 20 ]. Therefore, the identification of Sis1p is a convincing result and expands our knowledge on UPR-Cyto stress.

It shows that experimental gel-to-gel variation under these conditions highly exceeds biological variation. Such variation among technical replicates almost reaches the level of variation between different biological states i. The latter was not recognized as the overexpressed protein due to overloading and poor resolution, which resulted in overlaped protein spots in corresponding 2D gel area see Figure 2 B, spot h.

It is unclear why the well-shaped Kar2p spot showed the lower fold change, but it also seems incorrect result, because all other quantitative analyses including immunobloting described below showed considerably higher fold change values. Finally, abovementioned basic Gpm1 protein spot with false overexpression at standard protein load in IPG here showed an opposite result — repression.

It once more confirmed that results in a basic zone of broad range IPG-based 2DE gels are unreliable. In summary, high protein load in pH 3—10 IPG strips was not suitable for the analysis of UPR-Cyto stress, therefore standard sample load is preferable for this method.

This protein is not abundantly expressed under normal growth conditions; therefore, Kar2p spot is underrepresented in the control sample at lower protein amount, resulting in imprecise quantitative comparison.

Possibly, in this case the result of 1x load experiment was also improved, because higher fold change of spot h is practically identical to earlier pH 4—7 IPG-based 2DE analysis. It seems worth to use the high protein load as optimal conditions for the routine analysis of yeast protein samples by NEPHGE-based 2DE technique, because it has only advantages over standard protein load.

To confirm the results of 2DE study, we have done immunoblots using commercially available antibodies against two overexpressed UPR-Cyto proteins Kar2 and Sis1. Kar2p showed the highest overexpression in UPR-Cyto stress, but determined fold change greatly varied from 1. Representative images of Western blot analysis of Kar2p and Sis1p expression in crude yeast lysates are shown in Figure 3. Alongside with the overexpressed main Kar2p form, immunoblot has also revealed an additional Kar2p band of slightly higher molecular weight in the cells expressing MeH protein, which induces UPR-Cyto Figure 3 B.

Most likely it was a precursor of Kar2 protein with uncleaved signal sequence. Therefore, we have included both bands in calculation of Kar2p expression fold change.

The results of three independent experiments showed 3. Western blotting using antibodies against Sis1p showed 1. Verification of proteomic results by immunoblot. Lysates were prepared from galactose-induced yeast cells of S. Lane M - prestained protein ladder with molecular weights of bands indicated at the left. The blots were probed with antibodies against yeast Kar2 and Sis1 proteins. GAPDH was used as loading control. General characteristics of both methods are briefly summarized in Table 4.

The examples given above illustrate the essential differences between two methods in Tables 1 and 2 : IPG 3—10 Invitrogen 2DE method is reliable only for the analysis of acidic proteins, whereas NEPHGE method produced acceptable results in entire pI range and was especially suitable for the analysis of basic proteins.

The results of differential expression proteomics experiment with UPR-Cyto stress confirmed that high sample load onto pH 3—10 IPG strips is unsuitable for studies of biological effetcs by 2DE. The comparison at optimal protein loading conditions see Table 1 for IPG and Table 2 for NEPHGE reveals very similar performance of both methods in acidic range with almost identical spot reproducibility and quality.

Overall variation of spot volume parameters is also similar in this case, as higher variation averages in IPG-based 2DE are compensated by lower SD values. The parameters of detected protein spots reproducibility, quality etc. It suggests that in the acidic zone both spot separation methods reached some optimal level, which results in similarly good parameters of resolved protein spots. It is worth to discuss this in more detail, because it opens new opportunities.

Detection of up to good quality spots in a single small 2D gel by using Coomassie staining with relatively low sensitivity is a promising result. Taking into account experimental procedure and protein detection method, it seems difficult to achieve similar result using a broad range IPG strips. Thus, the main problem of pH 3—10 IPG strips seems to be limited amount of total protein that can be resolved into good quality spots on 2D gel. In fact, there are several specific steps where the proteins are lost during IPG-based procedure.

Therefore, it suggests that effects observed with Invitrogen strips in our study may be inherent to all pH 3—10 IPG strips in general. NEPHGE procedure does not include in-gel rehydration step gels are casted and used fresh , and this could explain lower protein loss during 2DE. It should be noted that detection and analysis of large number of protein spots does not require the large amount of protein in the gel.

Actually, we did not find any proteomic study on yeast proteins where a large number of protein spots at least as high as in our study was analysed by IPG-based 2DE using Coomassie staining method. It is not clear why several times more sensitive protein detection methods are necessary if it is possible to detect the same thousand of proteins by simple Coomassie staining after loading much larger amount of protein mixture onto 1st dimension gel.

This would be convenient for both quantitative analysis and mass spectrometry protein identification. Usually it is possible to unambigously identify any protein spot visualised by Coomassie staining. Most likely, 2DE analysis of a whole proteome using Coomassie stain was not being used due to limited protein capacity of IPG strips. Inability to detect some less abundant acidic proteins by the NEPHGE-based method at standard protein load was easily solved by increasing the amount of total cell protein.

The essential drawback of the NEPHGE-based method in acidic protein analysis is disappearance of some differentially expressed protein spots. The examples here were Hsc82 and Hsp82 protein spots. However, NEPHGE-based method enabled identification of aforementioned basic differentially expressed Sis1 protein and this would compensate drawbacks in the acidic pI range. It was reported earlier that when using CIF, a whole class of proteins very basic proteins is lost, whereas when using AIF, a certain amount of each protein in a protein class very acidic proteins do not enter the gel [ 11 , 12 ].

Here is important to note that these specific problems are rather small if compared to the main drawback of a basic 2DE method itself. A lot of proteins do not enter any 2D gels at all. Usually a very few membrane proteins are detected by 2DE. Moreover, there are also other protein classes that are not presented on 2D gels.

If all proteins from whole cell lysates would enter 2D gels, MeH and MeN should be presented at microgram amounts. However, no traces of these proteins were observed in 2D gels using both 2DE methods Figures 1 and 2. It is evident that the loss of more than a half protein amount during 2DE procedure [ 21 ] is rather specific and a lot of proteins are totally lost from the samples. Taken together, there is no ideal technique for 2DE method, because all techniques have some drawbacks.

In our case, it seems the most efficient way to be the usage of large format NEPHGE gels for a broad range pH 3—10 analysis, whereas in the acidic range the analysis could be doubled by the narrow range IPG mini-gels pH 4—7 or pH 4. IPG 3—10 Invitrogen 2DE method is reliable only in analysis of acidic proteins, because in basic side of 2D gels the results are not reproducible; meanwhile, NEPHGE method is suitable in the entire pI range and especially efficient for the analysis of basic proteins.

In this study this was exemplified by identification of highly basic protein Sis1p overexpressed during UPR-Cyto stress in yeast cells. Overexpression of Sis1p was confirmed by immunoblot analysis. Nevertheless, the narrow range pH 4—7 IPG Invitrogen technique is a better method for the analysis of acidic proteins. Considering all the results derived from tested techniques, it seems the most efficient way is to use large format NEPHGE gels for a broad range pH 3—10 analysis, whereas in acidic range the analysis could be doubled by the narrow range IPG mini-gels Invitrogen.

The plasmids were used for the transformation of the S. Yeast culture media, growth and induction of S. The aim of this study was to directly compare the first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis 2DE method and evaluate their impact on the results of biological experiment.

The same platform including pI range pH 3—10 gradient and the gel length 7 cm mini-gels was used for both methods. The study was designed according to the main tasks: i to evaluate experimental variation in both 2DE techniques by running the same samples several times; ii to assess biological variation in protein expression during UPR-Cyto stress in yeast cells using both 2DE methods and thereby compare their efficiency in the concrete biological experiment.

The biological material was essentially the same as reported previously [ 17 ] except that here we have used only measles virus proteins for the expression in yeast i. Briefly, the UPR-Cyto stress was induced by the expression of MeH protein and the pattern of cellular proteins resolved by 2DE was compared to protein pattern from the control cells transformed with empty expression vector pFGG3.

In addition, the yeast cells expressing MeN protein, which does not induce cellular stress, were used as internal control in this study. Experimental variation in both 2DE methods was evaluated using the same samples from three experimental variants expressing MeH, MeN or control cells. This analysis was doubled by loading 1x and 2x amounts of protein samples standard and high load conditions, respectively.

Biological variation in cellular protein expression was assessed by performing independent experiments transformation of yeast cells with vectors, growing yeast cells, induction of viral protein expression, preparation of whole cell lysates and 2DE with subsequent gel staining and image analysis at standard 1x protein load. Fold changes for differentially expressed proteins were calculated from at least three independent experiments at standard conditions and the results are given in Table 3.

In addition, fold changes of the same protein spots were calculated from three replicas of one independent experiment at high protein load and the values were also included in Table 3 for comparison. In principle, all operations in both 2DE methods were performed in parallel except for 1st dimension electrophoresis IEF — isoelectric focusing.

Therefore, the results should be influenced only by differences in the 1st dimension techniques and this enables their direct comparison. An equal volume of glass beads was added and the cells were lysed by vortexing at high speed, 8 times for 30 sec, with cooling on ice for 10 sec followed by keeping 30 sec at room temperature between each vortexing. Samples were diluted with IEF buffer if necessary and equal protein concentrations were used for two-dimensional gel electrophoresis.

For high-load 2DE experiments, more concentrated samples were prepared by using less volume of denaturing IEF buffer. The mixture of carrier ampholytes and IEF gel solution composition was made according to Klose and Kobalz, [ 12 ]. Briefly, ampholytes of pH Accordingly, it gives wider separation zone at the pH Briefly, two gel solutions were cast in succession in a vertical device for preparation of the two-layered rod gels of the first dimension quantities sufficient for a total of eight rod gels : 1.

For complete polymerization, the gels of the first dimension were held at room temperature for 30 min and then kept in a damp chamber for additional 72 hr. The first-dimensional separation of proteins in the rod gels was performed in a vertical electrophoresis device according to the operating instructions of the manufacturer WitaVision.

After the termination of electrophoresis, the rod gels were carefully pushed out of the glass tubes onto plastic rails, and adaptation to the conditions of the second dimension was achieved by a series of three min equilibrations in a corresponding equilibration buffer containing 75 mM DTT, followed by equilibration in the same buffer with mM 2-iodoacetamyde. Briefly, the IPG strips and rod gels of the first dimension were gently transferred from equilibration and storage rails to the top of the stacking gel zones and covered with 0.

All 2D gels in this experiment were scanned with calibrated ImageScanner III GE Healthcare under the same settings: blank filter, transparent mode and dpi resolution. Then the image was resolved into separate 2D gel images and these were imported into 2D gel analysis software. Protein spots were detected automatically by setting the same parameters smooth, saliency and min area for all analysed 2D gels.

Artefact spots mostly near the boundaries of the gels were deleted manually in every 2D gel with detected spots. Then gels were matched in separate small groups of three gels e. Various comparisons and calculations of parameters were performed as indicated in the legends of Tables 1 , 2 and 3.

All 2D gels were divided into acidic and basic parts according to the position of known cellular proteins with near neutral pIs. The line of neutral pI 7. Then acidic or basic gel parts were selected and required calculations for acidic and basic protein spots performed as it is indicated in Tables 1 and 2. Differentially expressed spots were also analysed in internal control samples from cells expressing MeN protein. The expression level of differentially expressed protein spots indicated by arrows in Figures 1 and 2 was similar in both control and MeN expressing cells data not shown.

The protein identification was carried out at the Proteomics Center in the Institute of Biochemistry Vilnius, Lithuania by means of tryptic digestion and mass fingerprinting. Tryptic digestion was performed according to earlier described procedure [ 28 ]. See it in action. Zoom Rooms. Zoom Phone. Zoom for Home.

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