As in any biological experiment, the controls are as important, if not more, than the actual samples. There are multiple options, and researcher needs to choose the controls depending on the question they would like to ask. Continue reading “Recommendations for Normalizing Reporter Assays”
Welcome to the third installment of our series on cell-based assays; in this post we talk about treatment parameters for cell-based assays. Designed for the newbie to the world of cell-based assays, we have covered the topics of choosing your cell type and basic cell culture tips in the previous posts. In this post, we will discuss how decisions about test compound treatment: how much and how long can affect assay results and interpretation.
Continue reading “Considerations for Successful Cell-Based Assays III: Treatment Parameters”
Many applications require amounts of protein that cannot be obtained using a eukaryotic cell-free expression system. As an alternative, a prokaryotic system can be used when this need arises. The E. coli S30 T7 High-Yield Protein Expression System is designed to express up to 500μg/ml of protein in 1 hour from plasmid vectors containing a T7 promoter and a ribosome binding site. The protein expression system provides an extract that contains T7 RNA polymerase for transcription and is deficient in OmpT endoproteinase and lon protease activity. All other necessary components in the system are optimized for protein expression. This results in greater stability and enhanced expression of target proteins.The following references highlight the use of this system for a variety of unique applications:
Loh, E. et al. (2011) An unstructured 5′-coding region of the prfA mRNA is required for efficient translation. Nuc. Acids. Res. (online) Examines the effect of upstream codon sequence/length on the correct ribosome binding and translation initiation of the pfrA protein.
Mitsuhashi, H. et al. (2010) Specific phosphorylation of Ser458 of A-type lamins in LMNA-associated myopathy patients. J. Cell. Sci. 123, 3893–900 By creating a series of mutations in the protein lamin A, Akt1 phosphorylation sites were determined.
Halvorsen, E. et al. (2011) Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis. Microbiology 157, 387–97. S30 High Yield System was used to characterize the inhibitory effect of Txe toxin on protein expression.
Mo, P. et al. (2010) MDM2 mediates ubiquitination and degradation of activating transcription factor 3. J. Biol. Chem. 285, 26908–15. By using in vitro pull down experiments the researchers characterized the binding of AFT3 to MDM2 leading to the proteolysis of AFT3 system by ubiquitination.
Working with RNA can be a tricky thing…it falls apart easily, and RNases (enzymes that degrade RNA) are ubiquitous. Successfully isolating RNA and maintaining its integrity is critical, especially when sensitive downstream applications are used (e.g., RNA-Seq).
Good techniques for RNA handling are simple to employ but crucial for success. All RNA purification and handling should take place in an RNase-free, RNA-only zone of the lab. Segregating RNA work from protein and DNA purification and handling will help minimize the potential for RNase contamination and help keep your RNA intact. Only buffer and water stocks treated to be RNase-free should be kept in the RNA area of the lab, and gloves should be worn at all times to prevent accidental contamination. Tools and equipment such as pipets, tips, and centrifuges should be designated for use only in the RNA zone as well. The location of the RNA zone in the lab is also important. Keeping traffic to a minimum and moving the RNA zone away from doors, windows, and vents can also help minimize contamination.
Using an RNase inhibitor can also help safeguard your samples from RNase degradation. These inhibitors can bind to any RNases that may have been introduced into your sample and prevent them from cutting the RNA present.
This is the second in a series of blog posts covering topics to consider when designing and performing cell-based assays. In the first installment, we discussed the importance of choosing the right cell type for your assay. Here we will discuss how cell culture conditions affect cell-based assays.
Continue reading “Considerations for Successful Cell Based Assays II: Cell Culture Conditions”“I would do more with my samples, but it’s just not possible…I know there’s probably a wealth of information in there, but there is just no way to get it out…I’ve got blocks of tissue sitting in the lab, experiments I want to run, but no good way to get clean nucleic acids out.”
These are a few of the comments I heard when talking with scientists at the American Society of Human Genetics meeting last week in Montreal. They, and countless other researchers, are sitting on a treasure trove of information that may have been locked away a few months ago, a few years ago, or decades ago. I’m referring to formalin-fixed, paraffin-embedded (FFPE) tissue blocks. It is estimated that there are upwards of 400 million tissue blocks archived globally and scientists are clamoring to find ways to best utilize nucleic acids derived from these tissues in applications like qPCR, microarrays, and next generation sequencing.1 Continue reading “Fixed in the Past, Focus on the Future”
mRNAs commonly exhibit differing salt requirements for optimal translation. Small variations in salt concentration can lead to dramatic differences in translation efficiency. The Flexi® Rabbit Reticulocyte Lysate System allows translation reactions to be optimized for a wide range of parameters, including
Mg2+ and K+ concentrations and the choice of adding DTT. To help optimize Mg2+ for a specific message, the endogenous Mg2+ concentration of each lysate batch is stated in the product information included with this product.
The following references utilize the features of Flexi Rabbit Reticulocyte Lysate System to investigate certain parameters of translation:
Vallejos, M. et al. (2010)The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation. Nucl Acids Res. 38, 618–32. Identification of internal ribosomal ribosomal entry site in the 5’ untranslated region of the mouse mammary tumor virus mRNA.
Spriggs, K. et al. (2009) The human insulin receptor mRNA contains a functional internal ribosome entry segment. Nucl. Acids. Res. 17, 5881–93. Identification of a functional internal ribosome entry site in the human insulin receptor mRNA.
Powell, M. et al. (2008) Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein. RNA 14, 2394–06. Analysis of the mRNA signals involved in the expression of influenza B virus BM2 protein.
Sato, V. et al. (2007) Measles virus N protein inhibits host translation by binding to eIF3-p40. J. Vir. 81, 11569–76. Charaterized the effect of the measles virus N protein binding to the translation initiation factor eIF3-p40 on the expression of various proteins in rabbit reticulocyte lysate.
Hirao, K. et al. (2006) EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J. Biol. Chem. 281, 9650–58. The EDEM3 protein was expressed in the presence of canine microsomal membranes to establish that co-translational translocation occurs into the endoplasmic reticulum.
Shenvi, C. et al. (2005) Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations–implications for the study of ribosome dynamics. RNA 11, 1898–08. Characterization of ribosome dynamics under different ionic conditions.
Nair, A. et al. (2005) Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary. J. Biol. Chem. 280, 42809–16. Examined the affect of luteinizing hormone receptor mRNA binding protein on transltional suppression of luteinizing hormone receptor RNA.
Detection of protein expressed using cell-free systems is required for most applications such as protein:protein interaction and protein:nucleic acid interaction studies. Traditionally, one adds radioactive [35S]methionine to cell-free expression reactions, and the methionine is incorporated into the expressed protein, allowing detection by autoradiography. Many researchers are moving away from radioactivity. Traditional Western blot analysis provides the researcher a nonradioactive method for detection but, if performed improperly, can result in high background, which can mask expressed proteins and affect downstream applications.
One critical step in producing low-background, high-signal Western blots is choosing the correct dilution of the primary antibody. Typically the manufacturer recommends antibody dilution from 1:1,000 to 1:2,500 for standard western blotting experiments. However when using crude lysates as a source of the target protein, these recommendations exhibit significant background. When the antibody was diluted 1:50,000, background was decreased significantly, and the positive signal was a large percentage of the total signal.
As a general recommendation when performing Western blot analysis of proteins expressed in cell-free systems, one must experimentally determine the optimal dilution of the primary antibody. In the Western blots performed in this study, primary antibodies were diluted ~50-fold more than the provider’s recommended dilution.
For additional technical details refer to this recent article published in Promega’s PubHub:
Hook, B and Schagat, T. (2011) Non-Radioactive Detection of Proteins Expressed in Cell-Free Expression Systems Promega Corporation Web site. Accessed August 17, 2011.
Why do few of my pGEM®-T or pGEM®-T Easy Vector clones contain the PCR product of interest?
There are several possible reasons why the PCR product may not be recovered after ligation, bacterial transformation and plating when using the pGEM®-T or pGEM®-T Easy Vector Systems.
The PCR fragment may not be A-tailed. Without the A overhangs, the PCR product cannot be ligated into a T vector. Use a nonproofreading DNA polymerase like GoTaq® DNA Polymerase for PCR. If a proofreading DNA polymerase is used, A overhangs will need to be added. Purify the PCR fragment, and set up an A-tailing reaction (see the pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual #TM042). The A-tailed product can be added directly to the ligation as described in the pGEM®-T or pGEM®-T Easy Vector protocol.
The insert:vector ratio may not be optimal. The ideal ratio for each insert to a vector can vary. For example, the Control Insert DNA works well at a 1:1 ratio, but another insert may be ligated more efficiently at a 3:1 ratio. Check the integrity and quantity of your PCR fragment by gel analysis. Optimize the insert:vector ratio (see Technical Manual #TM042).
Multiple PCR products were amplified and cloned into the pGEM®-T or pGEM®-T Easy Vector. Other amplification products including primer dimers will compete for ligation into the T vector, decreasing the possibility that the desired insert will be cloned. To minimize other competing products, gel purify the PCR fragment of interest.
Promega Technical Services Scientists are here to assist you in troubleshooting your experiments at any time. Contact Technical Services.
Analyzing more than one cellular biomarker (multiplexing) in a single sample is advantageous for a number of reasons. Multiplexing allows researchers to save money and time, while conserving critical samples. In addition, understanding the relationship between cell biomarkers can provide a more complete picture of cell health, leading to improved predictive models for drug discovery. Understanding biomarker relationships can also minimize ambiguity in the data set and validate if a treatment effect is real or an artifact of the system. To avoid repeat experiments and extract the most physiologically relevant data from multiplex cell-based assays, we discuss considerations around assay choices, cell type, cell culture, treatment parameters, detection and appropriate experimental controls.
Continue reading “Tips for Multiplex Cell-Based Assay Success”