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 …
Declarações
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