Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in Western blot analysis of leukocyte subpopulations from children

To study differences in the development of immunity, leukocytes from cord blood are often compared with those from adult peripheral blood. Western blot analysis is a common method for detecting proteins. In this study, we investigated the reliability of using different housekeeping proteins (β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) as internal controls for different leukocyte subpopulations from infants, children, and adults. Our results showed that the expression levels of β-actin and β-tubulin were much lower in cord blood leukocytes than in adult leukocytes, and this expression pattern persisted in children up to 3 years old. Further study revealed that the β-actin expression level in newborns was especially lower in CD14-positive monocytes. However, cord blood and adult peripheral blood monocytes had similar expression levels of β-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low β-actin expression level in neonatal monocytes. Thus, researchers should carefully assess the appropriate use of housekeeping gene-encoded proteins as internal standards to normalize samples for comparisons of different leukocyte populations from subjects of different ages. In this study, we determined that GAPDH was a more reliable internal control than others in Western blot analysis for comparing the development of immunity among infants, children, and adults.     

Housekeeping proteins: How useful are they in skeletal muscle diabetes studies and muscle hypertrophy models?

The use of Western blot analysis is of great importance in research, and the measurement of housekeeping proteins is commonly used for loading controls. However, Ponceau S staining has been shown to be an alternative to analysis of housekeeping protein levels as loading controls in some conditions. In the current study, housekeeping protein levels were measured in skeletal muscle hypertrophy and streptozotocin-induced diabetes experimental models. The following housekeeping proteins were investigated: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, α-tubulin, γ-tubulin, and α-actinin. Evidence is presented that Ponceau S is more reliable than housekeeping protein levels for specific protein quantifications in Western blot analysis.     

V3 Stain-free Workflow for a Practical,Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable.     

β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients

Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R2=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder.     

Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition

The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (β-tubulin and β-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ-COP). 2D-Western blot analysis of β-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells.     

草鱼MYH mRNA表达量分析中采用的内参基因稳定性比较

本研究分别以β-actin、18S rRNA和GAPDH为内参基因,采用实时荧光定量PCR对草鱼早期发育时期肌球蛋白重链(myosin heavy light,MYH)基因的mRNA表达量进行分析,并比较不同内参基因对MYH基因mRNA表达水平检测结果的准确性.研究结果表明,以β-actin和GAPDH作为内参,MYH基因mRNA表达水平完全一致,其表达量从原肠到仔鱼阶段逐次递增,仔鱼与原肠期阶段相比表达量差异显著;当采用18S rRNA作为内参时,MYH基因mRNA在发育阶段的表达量呈不稳定状态.因此,β-actin和GAPDH均可作为内参基因,用于草鱼早期发育中MYH基因mRNA的相对定量研究:而18S rRNA作为内参时,可能会对检测结果造成偏差.本研究不仅准确的揭示了草鱼MYH基因mRNA的表达特征,并且为荧光定量PCR技术在鱼类基因表达研究方面提供了有价值的参考.     

GAPDH is not regulated in human glioblastoma under hypoxic conditions

Background

Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results

We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion

GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.     

茶树实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是提高实时荧光定量PCR分析（qRT-PCR）准确性的先决条件。该文以茶树（Camellia sinensis）芽、叶、幼根、嫩茎、花瓣、种子和愈伤组织为材料,应用实时荧光定量PCR技术,分析了18S rRNA、GAPDH、β-actin和α-tubulin4个常用内参基因在茶树不同器官组织中的表达情况。经GeNorm和NormFinder软件分析发现,当利用荧光定量PCR分析比较茶树不同器官组织中的基因表达差异时,可选择β-actin作为校正内参基因;而比较不同成熟度的叶片和愈伤组织时,可以选择GAPDH作为校正内参基因。     

Stain-Free total protein staining is a superior loading control to β-actin for Western blots

Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots.     

Decreased expression of cardiac sarcoplasmic reticulum Ca2+-pump ATPase in congestive heart failure due to myocardial infarction

Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca²⁺-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca²⁺-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca²⁺-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.     

兔实验性动脉粥样硬化和急性心肌梗死双模型的建立

目的建立兔实验性动脉粥样硬化和心肌梗死双模型,比较血管新生在动脉粥样硬化和缺血心肌中发生机制的差异。方法选择20只雄性新西兰兔,随机分为两组,A组10只为普通饮食对照组,B组10只为高脂饮食组,共喂养9周。第3周末心导管封堵冠状动脉血管致急性心肌梗死。测定不同时期血脂水平。实验终点,苏丹Ⅲ染色测定主动脉斑块阳性面积;免疫组化染色测定不同心肌区域和主动脉血管壁CD34阳性反应强度,测定不同心肌区域新生血管密度;Western blot检测hypoxia-inducible factor1α(HIF-1α)在动脉粥样硬化和缺血心肌中的表达。结果高脂组血脂水平进行性增高。高脂组主动脉斑块阳性面积高于对照组,差异有显著性。在心肌正常区、梗死区和梗死边缘区:CD34阳性反应强度和新生血管密度各组间差异有显著性,HIF-1α的表达各组间差异有显著性;均为梗死边缘区最高,梗死区次之,正常区最低。在高脂组和对照组主动脉:CD34阳性反应强度两组间差异有显著性,HIF-1α的表达两组间差异有显著性;高脂组强于对照组。结论成功建立兔实验性动脉粥样硬化和心肌梗死双模型,提示动脉粥样硬化和缺血心肌中均有血管新生的参与。     

Relation between expression of TNF alpha, iNOS, VEGF mRNA and development of heart failure after experimental myocardial infarction in rats.

An injury to the heart due to myocardial infarction (MI) may progress to heart failure. Among factors, whose interactions promote remodeling of ischemic myocardium, the increased expression of tumor necrosis factor alpha (TNFalpha), inducible nitric oxide synthase (iNOS) and Vascular Endothelial Growth Factor (VEGF) was found. However, little is known about the temporal and spatial relation between expression of iNOS, cytokine TNFalpha, and growth factor VEGF during pathological process of development of heart failure after the myocardial infarction. Male Sprague-Dawley rats were used for experimental myocardial infarction. The procedure was performed by anterolateral thoracotomy and snearing LAD with the metal clip. The hemodynamic measurements were done with the Langendorff preparation converted into a working heart system. The hemodynamic parameters were recorded at day 6, 11, 28, 40 and the myocardium for gene expression was collected at day 1, 4, 11, 28, 40. Control group was sham operated rats. The VEGF, TNFalpha, iNOS, and GAPDH genes were detected by RT-PCR assay from samples taken at border zone of myocardial infarction. Expression of isoform VEGF120 was found at day 1 and 4 after MI, whereas isoforms VEGF164 and VEGF188 along with expression of TNFalpha and iNOS was found at day 1, 4, 11, 28, 40. No expression of examined genes was detected in the myocardium of control rats. The expression of studied factors was parallel with development of heart failure after myocardial infarction assessed by hemodynamic measurements. These findings confirm the postulated involvement of TNFalpha, iNOS and growth factor VEGF in the remodeling of the myocardium and development of heart failure after experimental myocardial infarction.     

人β-actin蛋白原核表达及其单克隆抗体制备

为获得分泌抗人β-actin蛋白单克隆抗体（McAb）的杂交瘤细胞,通过在大肠杆菌中原核表达人β-actin蛋白,以纯化的人β-actin蛋白作为抗原免疫BALB/c小鼠。经过细胞的融合及筛选获得1株能稳定分泌抗人β-actin蛋白McAb的杂交瘤细胞,命名为2B4。采用间接ELISA和Western blot方法对McAb的特异性、稳定性和适用范围进行鉴定。结果显示：蛋白的相对分子质量为43 kDa,可溶于8 mol/L尿素;杂交瘤细胞上清的抗体效价为1×10^5,腹水的抗体效价为1×10^7;间接ELISA结果表明,杂交瘤细胞在体外传20代或液氮冻存3个月后,分泌的抗体效价不变;37℃保存24 h后,抗体的效价开始下降。Western blot结果显示,单克隆抗体识别人、鼠、兔和鱼的β-actin蛋白,与其发生特异性反应。2B4分泌的单克隆抗体可以广泛的应用于细胞生物学和免疫学试验,具有良好的应用价值。     

鳜鱼基因表达转录分析中的内参选择比较

目前基因表达的转录分析多采用单一或多个看家基因作为内参来校正目的基因的表达量。该实验以鳜鱼6个不同组织和5个不同胚胎发育阶段为研究对象,应用实时荧光定量PCR技术,观察了GAPDH、β-actin和18S rRNA三个看家基因mRNA水平的表达情况。geNorm统计分析表明,胚胎发育阶段β-actin表达最为稳定;不同的组织样品间,GAPDH表达最为稳定;而18S rRNA 的表达在不同的发育阶段不稳定。当利用多基因作为内参时,使用两个最稳定表达的看家基因即可对目的基因的表达进行准确校正。该结果证实了基因表达转录分析中内参基因选择的必要性,同时为鳜鱼等鱼类基因表达分析时内参基因的选择提供有价值的参考