Protein purification and analysis: next generation Western blotting techniques

Introduction: Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results.

Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance.

Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.     

电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响

探讨了电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响.采用抗凋亡抑制蛋白-Survivin的抗体,对细胞裂解液进行蛋白质印迹.与常规操作方法不同之处是：在电转移的凝胶“三明治”中,重叠放置两张硝酸纤维素膜.电转移后,对两张膜同时进行免疫印迹.在特定的转移条件下,两张膜的免疫印迹都出现了Survivin蛋白的特异印迹带,证实了电转移中存在着蛋白质的透膜现象.转移时间、电流强度和蛋白质的分子质量,都是影响蛋白质透膜的相关因素.电转移中蛋白质的透膜,可以产生“印迹复制失真”的效应,从而最终影响蛋白质印迹定性和定量的结果.所得实验结果和结论,揭示了蛋白质印迹技术方法学中一个需要加以充分关注的问题,对于科学掌握和应用该技术具有积极作用.     

高灵敏的蛋白质免疫印迹新方法用于β-淀粉样蛋白检测

目的:对抗体进行改进,降低传统蛋白质免疫印迹方法的检出限。方法:在传统蛋白质免疫印迹方法的基础上,将一抗和二抗分别与纳米金和氧化石墨烯结合,以提高其对痕量样本的检测灵敏度,降低检测限。结果:优化后的方法对β-淀粉样蛋白的检测限从原来的432 pg/mL降低至16 pg/mL,为原来的1/27。结论:改进后的蛋白质免疫印迹新方法性能稳定,重复性好,可用于生物样本中痕量被检测物的测定。     

Heat Shock Induced Change in Protein Ubiquitination in Chlamydomonas

Ubiquitin was purified from pea (Pisum sativum L.) and its antibodywas produced. Western blot analysis showed that the antibodycross-reacted with ubiquitins from a green alga Chlamydomonasreinhardtii, a brown alga Laminaria angustata and a red algaPorphyridium cruentum but not with ubiquitin from a blue-greenalga Synechococcus sp. In Chlamydomonas, the antibody also reactedwith some ubiquitinated proteins including 28- and 31-kDa polypeptides.The isoelectric points of Chlamydomonas ubiquitin and the 28-and 31-kDa ubiquitinated proteins were 8.0, 8.9 and 10.3, respectively.The ubiquitinated proteins, including the 28- and 31-kDa polypeptideswere detected after in vitro ATP-dependent ubiquitination ofChlamydomonas cell extract with ^l25I-labeled bovine ubiquitin.Heat treatment of Chlamydomonas cells (>40°C) causeddrastic increase of ubiquitinated proteins with high mol wt(>60kDa), and coordinated redistribution or decrease of otherubiquitinated proteins and free ubiquitin. Quantitative analysisrevealed that the 28- and 31-kDa ubiquitinated proteins showeddifferent responses against heat stress, i.e. the former beingmore sensitive than the latter. (Received July 10, 1988; Accepted October 4, 1988)     

Accumulation of ubiquitinated proteins in mouse neuronal cells induced by oxidative stress

Ubiquitin protein conjugates are commonly detected in neuronal brain inclusions of patients with neurodegenerative disorders. The failure to eliminate the ubiquitin-protein deposits in the degenerating neurons may result from changes in the activity of the ubiquitin/ATP-dependent proteolytic pathway. This proteolytic pathway plays a major role in the degradation of short lived, abnormal and denatured proteins. Cadmium is a potent cell poison and is known to affect the ubiquitin pathway and to cause oxidative stress. Increases in protein mixed-disulfides (Pr-SSG) and decreases in glutathione (GSH) are often used as markers of oxidative stress. To investigate the relationship between the ubiquitin pathway and cellular glutathione (GSH), we treated HT4 cells (a mouse neuronal cell line) and rat mesencephalic primary cultures with different concentrations of the heavy metal. We observed marked increases in Pr-SSG as well as decreases in GSH, after exposure of HT4 cells or primary mesencephalic cultures to Cd²⁺. Furthermore, our results show that Cd²⁺ induced the accumulation of ubiquitinated proteins. Detection was by Western blotting of total cell extracts probed with antibodies that recognize ubiquitin-protein conjugates. These results suggest that the ubiquitin-pathway is closely involved in the cell response to cadmium-mediated oxidative stress. Abbreviations: GSH – glutathione; GSSG – glutathione disulfide; Pr-SSG – protein mixed disulfides.     

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.Download video file.^{(47M, mov)}     

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.     

Western blot analysis of cereal grain prolamins using an antibody to carboxyl-linked indoleacetic Acid

A monoclonal antibody raised against carboxyl-linked IAA was used in Western blot analysis of storage proteins from kernels of Avena sativa, Pennisetum americanum, Sorghum bicolor, and Zea mays. IAA or an IAA-like molecule is associated with the ethanolsoluble protein fraction of the seed. Western blotting of commercial zein, the major storage protein of maize, along with physicochemical evidence reported by Leverone et al. ([1991] Plant Physiol, 96: 1070-1075) indicated that IAA is linked with this prolamin. Results suggest that an IAA-prolamin association may be widespread throughout the Poaceae.     

Western blotting via proximity ligation for high performance protein analysis

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor β by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.     

Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting

We noted differences in the antibody response to 3-nitrotyrosine (NO(2)Tyr) in fixed and non-fixed tissues, and studied therefore potential problems associated with non-fixed tissues in Western blot analyses. Three different monoclonal anti-nitrotyrosine antibodies in Western blot analysis of inflammatory stimulated rat abdominal, liver and lung tissue homogenates caused no immunoreactivity, in contrast to a polyclonal nitrotyrosine antibody applied in fixed and non-fixed tissues. Western blot studies using both mono- and polyclonal antibodies showed a temperature- and heme group-dependent reduction of NO(2)Tyr in nitrated rat and bovine serum albumin incubated with dithiothreitol. Mass spectrometric analyses of a nitrated peptide angiotensin II revealed under similar conditions a positive temperature effect between 56 and 70 degrees C on reduction of NO(2)Tyr to 3-aminotyrosine which is not detected by anti-NO(2)Tyr antibodies. Western blot analysis may therefore underestimate the level of tissue nitration, and factors causing a reduction of NO(2)Tyr during sample preparation might conceal the actual nitration of proteins.     

Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.     

Detecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and hydrogen peroxide

Western blot detection of multiple proteins is challenged by the need to use antibodies from the same species and the harsh stripping methods that can remove protein or reduce protein antigenicity. Quenching using 27% hydrogen peroxide was developed as an alternative to stripping to inhibit horseradish peroxidase used to detect secondary antibodies. To detect two epitopes with same-species primary antibodies, quenching was followed by incubation in a precomplexed mixture of primary and secondary antibodies for the second epitope plus serum from that species. Both methods will be valuable in specific detection of multiple proteins by Western blotting, and will save time, valuable samples, and reagents.     

Characterization of a ubiquitinated protein which is externally located in African swine fever virions.

An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located. Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections. This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated. This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions. The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid. The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded. The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector. Recombinant PIG1 was ubiquitinated in the presence of E. coli expressed UBCv1 in vitro. These results suggest that PIG1 may be a substrate for UBCv1. The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquitinated protein present in virions. Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein.     

Identification of human hsp-70 expression in stably transfected rodent cells by isoelectrofocus immunoblots.

The major heat-shock protein, hsp-70, is synthesized by cells from a wide variety of organisms in response to heat shock or other stresses. It is assumed that hsp-70 may have an important thermal protective function. To test this hypothesis directly, we have transfected rat fibroblast cells with appropriate expression plasmids containing a cloned human hsp-70 gene. Stable transfectants expressing the human hsp-70 gene product were identified by Western blot analysis. During the course of selecting successful transfectants, we found that when standard methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining were employed with the monoclonal antibody raised against the human hsp-70 antigen, we were unable to differentiate the human hsp-70 from the heat-inducible rat hsp-70. This was because the monoclonal antibody cross-reacts with the human and rat proteins, which have the same mobility in SDS-PAGE, and it is difficult to determine which protein is expressed. To improve the resolution of the Western blot technique, we performed additional immunoblot analysis of cellular proteins separated by slab gel isoelectrofocusing. Our study shows that the isoelectrofocusing technique, when combined with antibodies against hsp-70, gave a better resolution for the separation of exogenous human hsp-70 and the endogenous rat hsp-70 than the commonly used SDS-PAGE Western blot analysis. It provides a rapid and specific method to identify positive colonies that express the human hsp-70 gene in transfected rodent cells.     

Intrahepatic assembly of very low density lipoproteins: immunologic characterization of apolipoprotein B in lipoproteins and hepatic membrane fractions and its intracellular distribution

Monoclonal antibodies, prepared against rat apoB, were used to examine apoB structure in serum lipoproteins and characterize the forms and localization of apoB in liver membrane fractions and cultured hepatocytes. Of the several antibodies obtained, four, having separate epitopes, were characterized. Western blot analysis showed that three (DB11, F4, and LB14) antibodies recognized both apoBL and apoBS. One antibody (HB41) recognized only apoBL. This antibody showed unusual properties. Competition ELISA assays showed that the epitope recognized by HB41 was more effectively expressed on low density lipoproteins (LDL) compared to very low density lipoproteins (VLDL). In addition, treatment of lipoproteins with detergents and sulfhydryl reducing agents also increased the expression of the HB41 epitope. Since HB41 has been found to inhibit LDL binding to hepatocyte receptors, these data indicate that the HB41 epitope is located on the carboxy-terminal side of the apoBS junction (probably within the LDL receptor binding domain). Western blotting hepatic microsomal subfractions showed that in the rough and smooth microsomes, HB41 recognized only apoBL, while in the Golgi it recognized both apoBL and a protein having a molecular weight slightly smaller. In contrast, Western blotting with a polyclonal antibody known to recognize both apoBL and apoBS showed that, in rough and smooth microsomes, proteins in addition to apoBL and apoBS having molecular weights between 120,000 and 30,000 were recognized. These proteins, likely to be proteolytic fragments of apoB, were barely detectable in the Golgi. Additional biosynthetic studies show that the [35S]methionine-labeled proteins smaller than apoB were immunoprecipitated from the rough microsome subfraction. Pulse-chase experiments show that these are produced with the same kinetics as full-size apoBL and apoBS, indicating that they are not incomplete nascent chains. Finally, immunofluorescence microscopy was used to determine the localization of monoclonal epitopes. ApoB monoclonal antibodies that recognized exclusively apoBL (HB41) and apoBL and apoBS (DB11) produced an immunofluorescence pattern characteristic of the endoplasmic reticulum, but not the Golgi. These data suggest that, in cultured rat hepatocytes, the majority of both molecular weight forms of apoB are localized in the endoplasmic reticulum, the initial site of VLDL assembly. The additional finding that proteolytic fragments of apoB are enriched in the microsomal fraction suggests that if the proteolysis occurs during subcellular fractionation, immature apoB is susceptible to proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting

Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published “–omics” data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in ‘skewing’ of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these “control” proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.     

Mouse PSP94 expression is prostate tissue-specific as demonstrated by a comparison of multiple antibodies against recombinant proteins

Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model.