Protein concentration was assayed after extraction of bilateral hippocampal tissue protein supernatant with protein lysate. The sample volume for the initial determination of the internal reference was 6 μL per well, the electrophoresis current was kept as constant as possible, and the electrophoresis voltages and time were as follows: 20 V-10 min, 60 V-20 min, and 100 V-60 min, respectively. The polyvinylidene fluoride (PVDF) membrane was removed and placed in triethanolamine-buffered saline (TBS) with Tween 20 (TBS-T) for washing (5 min × 3 times) and was blocked at room temperature (23-26°C) for 1 h. After these steps, the membrane was incubated with primary at 4°C overnight. Then, the membrane was washed and incubated in secondary antibody for 1 hours at room temperature for 1 h. Immunoreactive bands were visualised using the Image Quant LAS 4000 imaging system (Fujifilm USA, Valhalla, NY, USA), and densitometric analyses were performed using Image Gauge analysis software (Fujifilm, USA). The brightness and contrast of the raw blots were equally adjusted across the entire image using Adobe Photoshop CS5 software (Adobe Systems, Ottawa, ON, Canada) to generate representative images [34 (link)].
Image gauge analysis software
Manufactured by Fujifilm
Sourced in United States
Image Gauge analysis software is a tool designed for precise measurement and analysis of digital images. It provides users with advanced features for accurately quantifying various parameters within the images.
Automatically generated - may contain errors
3 protocols using image gauge analysis software
1
Protein Concentration Quantification in Hippocampal Tissue
2
Tau Kinase Assay Protocol
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Brain tau kinase assay was done according to our previous protocol (Planel, Miyasaka et al. 2004 ) with some modifications. 1µg of full length recombinant tau (reference T1001-2, R Peptide, Watkinsville, GA, USA) was prepared in sample buffer containing 5µg of RIPA cortex lysate. This reaction was incubated for 60 minutes in triplicates for each temperature (37°C, 40°C and 42°C) and terminated by adding one volume of sample buffer (NuPAGE LDS; Invitrogen, Carlsbad, CA) containing 5% of 2-β-mercapto-ethanol, 1 mM Na 3 VO 4 , 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA), and heated for 10 min at 95 °C. A negative control without ATP was processed in parallel following the same procedure in duplicate for each temperature. Samples were analyzed by SDS-PAGE and western blotting as described above. Immunoblot was analyzed using anti tau PS404 antibody (see section antibodies) since this epitope is phosphorylated by most of the major tau of kinases (Planel 2002) . The signal was normalized to total tau. Immunoreactive bands were visualized using ImageQuant LAS 4000 imaging system (Fujifilm USA, Valhalla, NY, USA), and densitometric analyses were performed with Image Gauge analysis software (Fujifilm, Valhalla, NY, USA). Images levels were adjusted.
3
Tau Kinase Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain tau kinase assay was done according to our previous protocol (Planel, Miyasaka et al. 2004 ) with some modifications. 1µg of full length recombinant tau (reference T1001-2, R Peptide, Watkinsville, GA, USA) was prepared in sample buffer containing 5µg of RIPA cortex lysate. This reaction was incubated for 60 minutes in triplicates for each temperature (37°C, 40°C and 42°C) and terminated by adding one volume of sample buffer (NuPAGE LDS; Invitrogen, Carlsbad, CA) containing 5% of 2-β-mercapto-ethanol, 1 mM Na 3 VO 4 , 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA), and heated for 10 min at 95 °C. A negative control without ATP was processed in parallel following the same procedure in duplicate for each temperature. Samples were analyzed by SDS-PAGE and western blotting as described above. Immunoblot was analyzed using anti tau PS404 antibody (see section antibodies) since this epitope is phosphorylated by most of the major tau of kinases (Planel 2002) . The signal was normalized to total tau. Immunoreactive bands were visualized using ImageQuant LAS 4000 imaging system (Fujifilm USA, Valhalla, NY, USA), and densitometric analyses were performed with Image Gauge analysis software (Fujifilm, Valhalla, NY, USA). Images levels were adjusted.
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