Quantitative Western Blotting to Estimate Protein Expression Levels
Published: May 31, 2023
Abstract
Source: Huang, Y. T., et al. Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting. J. Vis. Exp. (2019).
The video demonstrates a total protein normalization-based western blotting technique for quantifying the target protein expression in a sample. By utilizing the total protein staining of the sample, a ratiometric analysis is performed to determine the relative expression level of the target protein.
Protocol
NOTE: This protocol has been optimized using standardized, commercially available kits and reagents in order to increase reproducibility (see Table of Materials). 1. Preparation of samples Protein extraction Transfer snap-frozen cell or tissue samples from -80 °C on dry ice, thaw on ice, and wash as required with ice-cold 1x phosphate-buffered saline (PBS). Avoid unnecessary freeze-thaw cycles as this will aff…
Representative Results
Figure 1. Western blots showing TPS and Smn protein levels in mouse tissues at two different ages. TPS and Smn protein for P5 (A) and 10-week-old (C) mice. (B, D) The fluorescence intensity of whole-lane TPS was calculated and is indicated (in arbitrary units). M: marker/protein standard; kDa: kilodalton; a.u.: arbitrary unit; P5: postnatal …
Açıklamalar
The authors have nothing to disclose.
Materials
| Fine-Tipped Gel Loading Tips | Alpha Laboratories | GL20057SNTL | |
| Halt Protease Inhibitor Cocktail, EDTA-free 100x 5mL | ThermoFisher Scientific | 78437 | |
| Handheld homogeniser | VWR Collection | 431-0100 | |
| iBlot 2 Gel Transfer Device | ThermoFisher Scientific | IB21001 | |
| iBlot Transfer Stack, PVDF, regular size | ThermoFisher Scientific | IB401031 | |
| Image Studio Lite | Licor | N/A | Free download from https://www.licor.com/bio/products/software/image_studio_lite/ |
| IRDye 800CW secondary antibodies | Licor | — | Select appropriate secondary antibody that is specific against host of primary antibody. |
| Micro BCA Protein Assay Kit | ThermoFisher Scientific | 23235 | |
| Novex Sharp Pre-stained Protein Standard | ThermoFisher Scientific | LC5800 | |
| NuPAGE 4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well | ThermoFisher Scientific | NP0323BOX | |
| NuPAGE LDS Sample Buffer (4X) | ThermoFisher Scientific | NP0007 | |
| NuPAGE MOPS SDS Running Buffer (20X) | ThermoFisher Scientific | NP0001 | |
| Odyssey Blocking Buffer | Licor | 927-40000 | |
| Purified Mouse anti-SMN (survival motor neuron) monoclonal antibody | BD Transduction Laboratories | 610646 | Is used extensively in the SMN/SMA literature and gives consistent results regardless of lot number |
| REVERT Total Protein Stain, 250 mL | Licor | 926-11021 | |
| REVERT Wash Solution | Licor | 926-11012 | |
| RIPA Lysis and Extraction Buffer | ThermoFisher Scientific | 89900 | |
| XCell SureLock Mini-Cell | ThermoFisher Scientific | EI0001 |
Etiketler
Bu Makaleden Alıntı Yapın
Quantitative Western Blotting to Estimate Protein Expression Levels. J. Vis. Exp. (Pending Publication), e21367, doi: (2023).