China
Africa
Explore chapters and articles related to this topic
Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
A problem in northern blotting is often sample degradation by RNases (both endogenous to the sample and through environmental contami-nation), which can be avoided by proper sterilization of glassware and the use of RNase inhibitors such as DEPC (diethylpyrocarbonate). The chemicals used in most northern blots can be a risk to the researcher, since formaldehyde, radioactive material, ethidium bromide, DEPC, and UV light are all harmful under certain exposures. Compared to RT-PCR, northern blotting has a low sensitivity, but it also has a high specificity, which is important to reduce false positive results. The advantages of using northern blotting include the detection of RNA size, the observation of alternate splice products, the use of probes with partial homology, the quality and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and reprobed for years after blotting.
Glossary of scientific and technical terms in bioengineering and biological engineering
Published in Megh R. Goyal, Scientific and Technical Terms in Bioengineering and Biological Engineering, 2018
Southern blotting is a technique for transferring electrophoretically resolved DNA segments from an agarose gel to a nitrocellulose filter paper sheet via capillary action; the DNA segment of interest is probed with a radioactive, complementary nucleic acid, and its position is determined by autoradiography.
No-stain protein labeling as a potential normalization marker for small extracellular vesicle proteins
Published in Preparative Biochemistry & Biotechnology, 2023
Anjugam Paramanantham, Rahmat Asfiya, Siddharth Das, Grace McCully, Akhil Srivastava
Western blotting is a key method for assessing protein expression, which helps understand physiological processes in the cell under normal and pathological conditions as well as can guide the identification of new therapeutics. Variations in signal strength are common errors that can result in erroneous interpretations. Lane-to-lane normalization, utilizing housekeeping proteins or total protein can be used to mitigate the variations. Housekeeping proteins like β-actin, GAPDH, and α or β tubulin are the obvious choice for this approach. However, this approach is not applicable in all conditions as there are studies that show that the expression of these proteins is not always consistent and can change under some inherent or non-inherent conditions.[25,29–31]
IL-1α and IL-1β as alternative biomarkers for risk assessment and the prediction of skin sensitization potency
Published in Journal of Toxicology and Environmental Health, Part A, 2018
Min Kook Kim, Kyu-Bong Kim, Kyungsil Yoon, Sam Kacew, Hyung Sik Kim, Byung-Mu Lee
After treatment, cells were harvested, washed with cold PBS, and then lysed with protein extraction solution (PRO-PREP) (iNtRON Biotechnology, Seongnam, Korea) at 4°C for 20 min, and centrifuged for 5 min at 10,500g. Protein quantitation was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, Ill., USA) of the supernatant from lysed cells. Cell samples for western blotting, prepared to contain 20 µg of protein per 10 µl, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked for 1 hr in a Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat dry milk or 5% bovine serum albumin (BSA), and then incubated overnight at 4°C with an appropriate primary antibody [β-actin, 1:1,000; IL-1β, 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); IL-1α, 1:200; IL-31, 1:500 (Abcam, Cambridge, MA, USA); TNF-α, 1:500; COX-2, 1:500 (Cell Signaling Technology, Danvers, MA, USA); iNOS, 1:500 (Novus Biologicals, Littleton, CO, USA); PGE2, 1:500 (Biorbyt, Cambridge, UK)]. The membranes were washed with TBST and incubated with an appropriate secondary antibody [anti-mouse, 1:10,000 (Novus Biologicals, Littleton, CO, USA) or anti-rabbit, 1:5000 (GeneTex, Irvine, CA, USA)] for 1 hr at room temperature 25°C ± 2°C. The membranes were washed with TBST, reacted with ECL solution (Luminata Forte western HRP substrate) (Merck Millipore, Darmstadt, Germany), and developed by using a Davinch Chemi Imager (Davinch-K, Seoul, Korea). The quantification of band intensities was computed densitometrically by using ImageJ software (NIH, Bethesda, MD, USA). The band intensities were normalized to expression of B-actin, with the band intensities of the control group set as 1.
Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus
Published in Preparative Biochemistry and Biotechnology, 2019
Jun Zhao, Hai-Yang Yu, Yu Zhao, Feng-Hua Li, Wei Zhou, Bin-Bin Xia, Zhi-Yuan He, Jason Chen, Guo-Tuo Jiang, Ming-Li Wang
Western blotting, also known as immuno-blotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells or tissue.