From dissected hemi-brains of adult mice, protein amounts averaged 2–3 mg from the cortex, 400–600 μg from the hippocampus, and 1–2 mg from the cerebellum. Examples of protein amounts from cell cultures are 50–100 μg from 2 × 10 5 hNPCs, 150–300 μg from 2 × 10 5 hNPCs differentiated into neurons for 28 days, and 15–25 μg protein from 1 × 10 5 mature astrocytes. The user may need to adjust the volumes if substantially larger or smaller amounts of cells/tissues need to be used. Using either protein extraction method should yield solutions with quantifiable amounts of protein. Use appropriate personal protective equipment (gloves, eye protection) when handling of these reagents. Review the SDS document for these reagents before use. We recommend keeping the kit reagents in a chemical safety hood or a closed container. Avoid contact with skin, eyes, and inhalation of vapors. Use appropriate personal protective equipment (gloves, eye protection) when handling these reagents.ĬRITICAL: The Revert™ 700 Total Protein Stain Kit components contain acetic acid (CAS# 64-19-7) and sodium hydroxide (CAS# ). We recommend keeping the kit reagents contained in a chemical safety hood or a closed container. Avoid direct skin contact and inhalation of vapors of methanol and Revert™ Kit reagents. Specific inquiries regarding membrane types other than nitrocellulose should be investigated with the manufacturer.ĬRITICAL: Methanol (CAS# 67-56-1) is required for the Revert™ 700 Total Protein Stain Kit. Note: PVDF membranes are not recommended for use with the Bio-Dot® Microfiltration Apparatus, as per the product manual. A template, such as a pipette tip rack, would be helpful for aligning the dots on the membrane in a uniform manner, as analysis in the ImageStudio™ software can be done using a 96-well template rather than individual selection of each dot ( Wehr and Levine, 2012). In this case, the user should take care to keep the membrane as clean as possible (the infrared detection system is more sensitive to background contamination than other methods such as chemiluminescence ( Schutz-Geschwender et al., 2004)). Similar vacuum apparatuses are available through other manufacturers ( Guillemin et al., 2009), the specific user manuals for these should be referenced for any necessary modifications.Īlternatively, dots can be applied freehand, as has been published by other laboratories (Au - Chunhui et al., 2018, Tizon et al., 2010). Details on setup and cleaning of the apparatus can be found in the product manual ( ). The current protocol is designed for use with the 96 “dot” array, but could be modified for the slot array ( Bronowicka-Szydełko et al., 2020). Adapters are available for the application of either 96 “dots” in a 12x8 array, or as rectangular “slots” in a 6x8 array. The Bio-Dot® Microfiltration Apparatus (Bio-Rad) is made of polysulfone with a silicone gasket, designed for the application of samples to a membrane using vacuum. The Bradford Assay (Bio-Rad, Cat# 5000201) can be used for protein quantification, but any other similar method could also be used. 1x TBS should contain 25 mM Tris and 0.15 mM NaCl, buffered to pH 7.6. If analysis of other protein fractions is desired, alternate buffer formulations can be used as appropriate. This buffer system is for the extraction of soluble native proteins from cells or tissues. These can be made in-house, or purchased commercially (as listed in the Key Resources Table). Tris-buffered saline (TBS) and TBS + 0.1% Tween-20 (TBST) are required. Similar cells can be purchased commercially or derived through other differentiation processes. IR-wavelength secondary antibodies are also available from other vendors and could potentially be utilized as well.įor generation of neural stem cells and neurons, iPSCs (cell line ND50031 from RUCDR Infinite Biologics) were differentiated using protocols commercially available from STEMCELL™ Technologies. Primary antibodies as appropriate for sample/targets of interest should be selected, and the appropriate IRDye® Secondary antibodies (LiCor) selected.
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