Western Blot Analysis

Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo salar using gel-electrophoresis and Western blot Goh Xin Rong and Lim Yi Fei M15606 11 August 2015 BL6404 Advanced Biochemsitry INTRODUCTION The separation of proteins through polyacrylamide gel electrophoresis (PAGE) is one of the fundamentals of proteomics. This molecular sieving analytical technique sets the stage for various research applications such as the study of protein expression levels as well as clinical applications in the identification of prognostic biomarkers and disease differentiators [1]. The basis for PAGE is that a charged molecule would migrate towards a terminal of opposite polarity in an applied electric field. In proteomics study, the samples are often denatured and treated with sodium dodecyl sulfate (SDS). This ensures that the protein samples will be uniformly charged. These gels are often termed as nonnative PAGE. Typically, most protocols specify the need of a stacking and resolving gel for SDS-PAGE. Unlike the resolving gel, the stacking gel has a larger pore size, made possible by its lower acrylamide content and lower pH, typically of pH 6.8. It also possesses a different ionic content. While the resolving gel is responsible for separating the proteins into their respective molecular weights, the stacking gel ensure that all the proteins will simultaneously run in a common band. This stacking effect is achieved through the interplay of two major ions - chloride and glycine [2].Owing to their high mass-to-charge ratio, chloride ions present in the stacking gel are highly mobile and would be the first to migrate towards the anode. On the other hand, glycine, which is present in the running buffer would be the last to migrate towards the anode. As a weak acid, glycine can exist either as an uncharged zwitterion at low pH or negatively charged glycinate anion at higher pH [3]. During SDS-PAGE, glycine migrates away from the cathode as they move towards the stacking gel. Given the lower pH in this layer, glycine is protonated and its movement is consequently retarded. The combined effect of these two ions generate a region of high electric field strength that propels the movement of the negatively charged denatured proteins. The stacking gel can be omitted when utilising a gradient gel given the continuously decreasing pore size rendered by the gel [4]. Alternatively, some researchers commonly utilise the native PAGE, or non-denaturing PAGE. The main differentiating factor between these two PAGEs lies-in that the proteins will only be separated by size in the non-native PAGE, while they will be separated by both the protein's native charge and hydrodynamic size as they migrate towards the positively charged anode. The greater the negative charge, the faster the protein would move. This is concurrently impeded by the sieving effect of the gel that retards the movement of larger and bulkier proteins. The protein’s native charge is dependent on its amino acid composition and subsequent pI in the running buffer. Given that the proteins will still be in their folded configuration, native PAGE provides the advantage of being able to study the effects of biochemical processes that alters protein configuration or involves protein aggregation. Besides, its non-denaturing properties enable researchers to recover or extract particular bands of the separated proteins for further analysis [5]. Usually, these proteins retain their active conformation and enzymatic functions [6]. However, only non-native PAGE provides the ability of molecular weight estimation through cross-comparisons with a ladder.

Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo salar using gel-electrophoresis and Western blot

Goh X.R. and Lim Y.F.

In this analytical study, the use of Western blot was closely followed. After transferring the separated proteins onto a suitable membrane, antibodies are designed to detect general of specific proteins. These membranes typically fall under a selection of nitrocellulose or polyvinylidene fluoride constituents. More recently, Immobilon™-FL PVDF Membrane by Sigma Aldrich has been optimised to increase sensitivity and fluorescence applications [7]. Experimental overview Four samples of pre-prepared extracted proteins from Saccharomyces cerevisiae, Torenia sp., Gallus gallus domesticus and Salmo salar were separated with SDS page and transferred onto a Polyvinylidene fluoride (PVDF) membrane. The amount loaded into the polyacrilamide gel for the SDS Page was unified at 20µg for each sample. The membrane was then treated with antibodies specific to HSP70 (70kD) and Tubulin (50kD) in a Western Blot, and finally was visualised via colorimetric method (with TMB solution) or chemiluminescent method. The colorimetric results were visualised from Xin Rong and Yi Fei’s PVDF membrane, whereas the chemiluminescent results were visualised from Nadia, De Rong and Jing Yee’s PVDF membrane. The Commassie Blue staining results for the polyacrilamide gel was included for comparison as well. The results for colorimetric visualisation revealed significant losses of proteins during running of SDS-PAGE and overloading of protein samples, whereas the chemiluminescent results showed a certain extent of protein sample loss. Both methods successfully visualised the separation of HSP70 and Tubulin from the protein extract of all four organisms. RESULTS Protein Ladder

Fig 1. Colorimetric result (Left) and Chemiluminescent result (right) for proteins extracted from plant, fish, chicken and yeast Organism Plant Fish Chicken Yeast

Colorimetric Chemiluminescent HSP 70 Tubulin HSP 70 Tubulin Very faint band Very faint band Faint band Very faint band Negative band Normal band Faint band Normal band Negative band Very faint band Faint band Very faint band Normal band Very faint band Fiant band Very faint band Table 1. Summary of Results fro HSP 70 (70kD) and Tubulin (50kD)

DISCUSSION

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Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo salar using gel-electrophoresis and Western blot

Goh X.R. and Lim Y.F.

The presence of bands in the 70kD and the 50kD region, although very faint for some, indicates that HSP 70 and the Tubulin proteins were successfully extracted and separated from the plant, fish, chicken and yeast protein samples. From this it can be assumed that the major steps of Western Blotting - the transfer of separated proteins from gel to PVDF membrane, the binding of antibodies to HSP 70 and Tubulin proteins, and the generation of coloured or luminescent products from enzymeconjugated antibodies were successful despite protein losses in the process. Below is the description for the Western Blot bands analysis for both visualisation methods Band Properties Very faint / Absent

Explanation In the colorimetric result, the bands in first lane (plant) was so faint that only the right edges were slightly visible. The last few bands of the ladder were also not visible. The chemiluminescent results also showed very faint bands for the first two lanes. This could be an indication of extremely low protein concentration. Cross-referencing with the Coomasie blue stain performed after the SDS Page revealed that the faint band was not a result of errors during transfer to membrane or antibody binding during western blotting. Since the protein samples has satisfactory yields as tested previously, and the samples were loaded in the same amount (20µg), we can conclude that the error arose right before the SDS Page was run, and not as a result of inefficient extraction in prior experimentations. A possible factor is the fact that the protein was left in the loading well for quite a while before the gel was run due to errors in syncing with other groups. This could have caused the protein samples to diffuse into the buffer solution before the gel was run. It provides an explanation for the fact that the first two lanes – the ladder and the plant proteins – were extremely faint whereas the last few lanes did not experience such an effect, likely because the first two lanes were loaded first and experienced more diffusion. This explains how across lanes, bands can range from indication of very high protein load (negative bands for HSP70 in Fish proteins) to little to no protein load (extremely faint bands for HSP70 in Plant proteins) despite the fact that we placed in the same amount (20µg). It was hypothesised that on top of the loading sequence, smaller protein molecules diffuse out of the loading well faster than larger molecules. This explains the absence of bands further down the ladder, and the faint bands for nearly all Tubulin proteins in contrast with the HSP70. The same problem occurred with the chemiluminescent results.

Extra bands

In the future, it should be clearly indicated that all SDS Pages need to run immediately after the sample is loaded, and for students to coordinate for that purpose. It was clear that although the antibody was meant for HSP 70 and Tubulin, more than two bands were observed in each protein sample lane, especially for the chemiluminescent result (the bands in colorimetric result were probably lost when smeared). Other than the non-specific binding of antibodies, this could be the result of sample degradation and protein overloading, which are more likely to be the case. Prevention include optimising the use of protease inhibitors in buffers, as well as placing less protein load in wells for protein samples with very high concentrations (>15µg/µl). 3

Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo salar using gel-electrophoresis and Western blot

Goh X.R. and Lim Y.F.

Table 2. Western Blot band analysis

COLORIMETRIC RESULTS ANALYSIS (Xin Rong, Yi Fei) On top of the issues above, the colorimetric results show severe smearing and frowning effect. Other than protein degradation, a likely factor for smearing is overloading of protein samples, which could be prevented by loading less proteins into the gel electrophoresis loading well. Issues with Western Blot bands specific to the chemiluminescent result are as follow: Band Properties Negative

Details For the chicken and fish proteins, the bands appeared white in the centre and coloured at the edges – a phenomenon known as negative bands. This was the result of substrate depletion of the chromogenic substrate (TMB solution) by the bound antibodies due to protein concentration being too high. Curiously this occurred only for fish and chicken despite all the lanes containing the same amount of proteins. To prevent this, one could possibly load less proteins for proteins with extremely high concentration relative to other samples, with disregard to the same protein load (20µg) as used in this experiment.

Frowning

The electrophoresis process was ran too quickly – as a result the protein samples at the edges of the band travelled faster than the main band, possibly due to sample diffusion into edges of loading wells which were not properly made. A solution would be to investigate the expected running time for a sample, and to adjust the voltage accordingly with reference to the progress of the solvent front movement in the gel. Table 2. Western Blot band analysis for Chemiluminescent Results

Furthermore, there were patchy protein stains near the ladder as well, which could either be attributed to uneven distribution of antibodies as a result of placing more than one membrane in a container during incubation. As a result, areas of strips in contact with other membranes may not have as much antiboties bound to the desired protein as compared to membranes developed alone in a container. What we could do is to incubate strips individually in smaller containers to achieve the purpose of saving antibodies and prevent the effect of uneven distribution at the same time. There were patchy protein stains near the ladder as well, which could either be attributed to uneven distribution of antibodies as a result of placing more than one membrane in a container during incubation. As a result, areas of strips in contact with other membranes may not have as much antiboties bound to the desired protein as compared to membranes developed alone in a container. What we could do is to incubate strips individually in smaller containers to achieve the purpose of saving antibodies and prevent the effect of uneven distribution at the same time. Another possible factor of the patchy protein stains could arise during the transfer of proteins from the polyacrimide gel to the PVDF membrane. While protein transfer from gel to membrane is a common source of error, such as unknown protein bltoches and loss of protein, the full profile of the protein successfully transferred onto the membrane is not available for investigation. Hence, to obtain quality transfers, the Ponceau Red dye could be used to visualise transferred proteins temporarily to 4

Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo salar using gel-electrophoresis and Western blot

Goh X.R. and Lim Y.F.

determine if the protein bands are sharp and distinct or patchy and uneven. Certain problems could arise when gels are transferred under high voltage and temperature, or when small proteins accidentally transfer through the membrane. With the Ponceau Red dye, the transfer time and voltage can be optimised for a better result.

CHEMILUMINESCENT RESULTS ANALYSIS (De Rong, Nadia, Jing Yee) The bands from this result were more defined, though the dye front was slanted. This was possibly due to bubbles present between the bottom of the gel and container, which affects the uniform transmission of electric field through the gel. There are no signs of overloading or the frowning effect. The separation and detection of Tubulin and HSP70 from the organisms’ protein extract was successful, but certain bands were faint as explained above. In general, the poor result from colorimetric detection arose from errors during protein loading in the SDS-PAGE process. Colorimetric detection in general are cost effective and simpler in procedure, whereby the alkaline phosphatase enzyme simply converts the soluble chromogenic substrate (TMB solution) into coloured insoluble products, which precipitates on the site of conversion and produce coloured bands on the membrane. Chemiluminescent detection on the other hand uses the horseradish peroxidase enzyme to convert the peroxide / luminol solution into luminescent substrates. As multiple exposures are necessary to capture optimal signals, it is more troublesome to use despite being more sensitive than the colorimetric detection method. It also cannot be multiplexed, ie. analyse several proteins in an assay and may not be quantitative. [8] ALTERNATIVES TO WESTERN BLOTTING Immunoassays remain important today as the preferred method to detect the presence and concentration of particular proteins. Western Blot remains highly specific as it separates molecules based on two parameters – physical size and charge, hence there is less chance for false positive or negatives. However, the procedures are relatively more complex, and more sample volumes are required for each run. Limitations such as time consuming procedure due to its low or medium throughput, and the fact that it requires proteins to be denatured made alternatives such as ELISA and Protein Assays desirable in occasions of large scale screenings. ELISA (enzyme-linked immunosorbent assay) uses solid phase immunoassays (EIA) to detect antigens in liquid samples. ELISA is capable of testing protein molecules in its native state, and are technically easier than Western Blot (thus posing a potential for automation). ELISA, like Western Blot, can only investigate a limited number of proteins at a time. When multiplex capability is needed, Protein Microarrays prove useful as they can analyse up to 9,000 proteins at a time, whereas ELISA can at most analyse 96 or 386 well plates, and for Western Blot, less than 10. Like ELISA, Protein Microarrays are useful for relative quantitation of proteins or in tests that require proteins to be in the native stage. The difference lies in the Protein Microarray’s low sample usage as well as the high throughput analysis. [9] However, when measuring protein-protein interactions, it is still the best to use Western blot, as ELISA and Protein Microarrays occasionally gives false positives or negatives. In short, the type of immunoassay used depends on the need and available resources.

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Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo salar using gel-electrophoresis and Western blot

Goh X.R. and Lim Y.F.

APPENDIX

Fig 2. Coomassie Blue staining for extracted proteins

REFERENCES [1] Ikonomov V, Melzer H, Nenov V, Stoicheva A, Stiller S and Mann H, 1999, Importance of sodium dodecyl sulfate pore-graduated polyacrylamide gel electrophoresis in the differential diagnostic of Balkan nephropathy, Artificial Organs 75-80, http://www.ncbi.nlm.nih.gov/pubmed/9950183 [2] Thermo Fisher Scientific, n.d., Tricine protein gels, http://www.thermofisher.com/sg/en/home/lifescience/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/tricineprotein-gels.html [3] Dyche Mullins, 2002, How does an SDS-PAGE gel really work, Mullins Lab, University of California, San Francisco http://mullinslab.ucsf.edu/sds-page/ [4] Thermo Fisher Scientific, 2010, Thermo Scientific Pierce Electrophoresis Technical Handbook, v2, https://tools.thermofisher.com/content/sfs/brochures/1601925-Electrophoresis-Handbook.pdf [5] Alliance Protein Laboratories, n.d., Native gels, http://www.ap-lab.com/native_gels.htm [6] Thermo Fisher Scientific, n.d., Overview of protein electrophoresis, https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierce-protein-methods/overview-electrophoresis.html [7] Sigma-Aldrich, n.d., Immobilon® PVDF Membranes, http://www.sigmaaldrich.com/lifescience/molecular-biology/molecular-biology-products.html?TablePage=14561752

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Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo salar using gel-electrophoresis and Western blot

Goh X.R. and Lim Y.F.

[8] Licor Bio Blog, Which Western Blot Detection Method Should You Use? http://www.licor.com/bio/blog/western-blotting-2/western-blot-detection-method-fluorescencechemiluminescence-and-colorimetric [9] Grace Biolabs, nd., Immunoassays: Protein Arrays vs. ELISA and Westerns http://www.gracebio.com/blog/protein-array-elisa-western/

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