Western blotting was carried out according to the manufacturer’s instructions and previous studies [30 (link), 31 (link)]. Cerebral cortex and hippocampus extracts were loaded onto 10-16% Tris/tricine SDS gels and transferred to nitrocellulose membranes prior to overnight incubation with one of the following primary antibodies: BACE, BACE1, sAPPβ, PS1, NCT, Aph-1α, Pen-2, p-Ser199, p-Ser202, p-Thr205, p-Thr231, p-Ser396, p-Ser404, HT7, p25, p35, and Cdk5 (purchased from Abcam, Cambridge, MA, USA). MAP1, SYP, and PSD95 were obtained from Cell Signaling Technology, Inc. USA. Mouse monoclonal anti-IL-1β, mouse monoclonal anti-MMP-2, mouse monoclonal anti-MMP-9, and goat anti-mouse IgG labeled with biotin were procured from Santa Cruz Biotechnology, Inc. USA. Rabbit anti-mouse β-actin was also obtained from Santa Cruz Biotechnology, Inc. USA. The optical densities of the specific bands were achieved by image analysis software (HPIAS 2000, Tongji Qianping Company, Wuhan, China).
P ser396
Manufactured by Abcam
Sourced in United States
P-Ser396 is an antibody that recognizes the phosphorylated form of the protein at serine residue 396. It can be used to detect and quantify the phosphorylation of this specific site.
Automatically generated - may contain errors
Lab products found in correlation
P ser199, by Abcam (3 mentions)
P thr205, by Abcam (2 mentions)
P thr231, by Abcam (2 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti mmp 2, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti mouse β actin, by Santa Cruz Biotechnology (1 mentions)
Pen 2, by Abcam (1 mentions)
Mouse monoclonal anti il 1β, by Santa Cruz Biotechnology (1 mentions)
Bace1, by Abcam (1 mentions)
Anti cdk5, by Abcam (1 mentions)
Anti gsk3β, by Abcam (1 mentions)
Anti p35p25, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Bca assay, by Boster Bio (1 mentions)
β actin, by Proteintech (1 mentions)
3 protocols using p ser396
1
Western Blotting of Alzheimer's Markers
2
Quantitative Analysis of Tau Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For reducing SDS‒PAGE analysis, cell or brain lysates were mixed with 4x Laemmli buffer containing 10% β-mercaptoethanol (BME) and boiled at 97 °C for 5 min. For nonreducing SDS‒PAGE analysis, cell or brain lysates were mixed with 4x Laemmli buffer without BME. For immunoblot analysis, 10 μg of each lysate was separated on a 10% SDS–PAGE gel and transferred to a PVDF membrane. The levels of total tau and phosphorylated tau were detected by anti-tau antibodies against 2B11 (IBL), Tau5 (Abcam), pSer199 (Abcam), pSer396 (Abcam), and pThr205 (Abcam). For immunoblot analysis of tau kinases, the levels of total and phosphorylated tau kinase were detected by anti-GSK3β (Abcam), anti-ERK1/2 (Cell Signaling), anti-P35/P25 (Cell Signaling), and anti-CDK5 (Abcam) antibodies. β-actin (Abcam) and GAPDH (Cell Signaling) were used as loading controls. Band intensity was quantified using ImageJ software (NIH). All data were normalized to β-actin.
3
Western Blot Analysis of Tau Phosphorylation
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The total protein was obtained with the cell lysis buffer for western and IP containing a protease and phosphatase inhibitor (Beyotime Inc., Shanghai, China). We collected the supernatants after centrifugation and then measured the concentration via a bicinchoninic acid (BCA) assay (Boster Biological Technology co.ltd., Wuhan, China).
The prepared protein samples were separated in 10% Bis-Tris SDS-polyacrylamide gels and then transferred to a nylon-PVDF Immobilon-PSQ membranes at 200 mA. The membranes were blocked in blocking buffer for 1 h at room temperature, and then were incubated overnight with primary antibody at 4C. The primary antibodies were included Tau5 (1:5000, Abcam, Cat # ab80579), p-Ser199 (1:5000, Abcam, Cat # ab81268), p-Ser396(1:5000, Abcam, Cat # ab109390), p-Thr231(1:5000, Abcam, Cat # ab151559) and β-actin(1:10000, Proteintech, Cat # 66009-1-Ig). After washing for several times, the membrane was incubated with secondary antibodies (1:7500; Proteintech, Cat # SA00001-1 and SA00001-2) for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection (Biosharp, Beijing, China), and were scanned using GelView 6000 Pro (antpedia, China). Band density was measured using Image J software.
The prepared protein samples were separated in 10% Bis-Tris SDS-polyacrylamide gels and then transferred to a nylon-PVDF Immobilon-PSQ membranes at 200 mA. The membranes were blocked in blocking buffer for 1 h at room temperature, and then were incubated overnight with primary antibody at 4C. The primary antibodies were included Tau5 (1:5000, Abcam, Cat # ab80579), p-Ser199 (1:5000, Abcam, Cat # ab81268), p-Ser396(1:5000, Abcam, Cat # ab109390), p-Thr231(1:5000, Abcam, Cat # ab151559) and β-actin(1:10000, Proteintech, Cat # 66009-1-Ig). After washing for several times, the membrane was incubated with secondary antibodies (1:7500; Proteintech, Cat # SA00001-1 and SA00001-2) for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection (Biosharp, Beijing, China), and were scanned using GelView 6000 Pro (antpedia, China). Band density was measured using Image J software.
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