Anti-Drf1 and anti-Dbf4 were used at concentrations of 1/10000 and 1/3000 respectively in PBS with 0.1% Tween and incubated overnight. at 4°C. Anti-HA, anti-Myc and anti-βTrcp were used at a concentration of 1/1000 in PBS+0.1%Tween and incubated 1 hr at room temperature. Anti-Chk1 was used at a concentration of 1/100 in PBS+0.1%Tween and incubated 1 hr at RT. Goat anti-rabbit (Thermo Scientific 31466) and horse anti-mouse (Vector Laboratories PI 2000) HRP coupled antibodies were used as secondary antibodies and used at concentrations of 1/10000 in PBS+0.1%Tween and incubated for 1 hour at room temperature.
Pi 2000
Manufactured by Vector Laboratories
Sourced in United States
The PI-2000 is a laboratory equipment product manufactured by Vector Laboratories. It is designed to perform a core function within the laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Pi 1000, by Vector Laboratories (10 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Ab53004, by Abcam (2 mentions)
Ab18723, by Abcam (2 mentions)
Ab108539, by Abcam (2 mentions)
Pi 9500, by Vector Laboratories (2 mentions)
A5441, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Ab24170, by Abcam (1 mentions)
Ab18180, by Abcam (1 mentions)
Protease phosphatase inhibitor, by Cell Signaling Technology (1 mentions)
Bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 filter, by Merck Group (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Ab110289, by Abcam (1 mentions)
25 protocols using pi 2000
1
Immunoblotting antibody incubation conditions
2
TRPV1 Expression in Subcellular Fractions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
vHip samples underwent subcellular fractionation protocols as in [33 (link)]. Cellular and membrane samples (50 µg/subject) were then subjected to SDS-PAGE and Western Blot protocols using TRPV1 primary antibody (Thermo Fisher PA1-29421; 1:1000). Primary antibodies for Beta-actin (Thermo Fisher PA1-183; 1:1000) and Na+/K+ ATPase (MilliporeSigma 05-382; 1:1000) and associated secondaries (Vector PI-1000 and PI-2000; 1:1000) were used as respective loading controls for cellular and membrane samples. Optical density of all bands was imaged and quantified using ChemiDoc XRS (Bio-Rad) and Image Lab software. Bands were normalized by calculating the ratio of TRPV1 to loading control optical density for each animal. All data reflect the average of samples run in duplicate.
3
Quantitative Western Blot Analysis of Cholesterol Regulators
4
Antibody Validation for Cellular Protein Detection
5
Western Blot Analysis of Mitochondrial Proteins
6
Hippocampal Microglial Activity and Neurogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess microglial activity within the hippocampus, immunohistochemical staining was performed using rabbit anti-Iba-1 polyclonal antibodies (1:500, ab108539) and anti-CD86 rabbit monoclonal antibodies (1:1000, ab53004) (Abcam, Cambridge, MA, USA). To assess neurogenesis intensity, the number of newly formed neurons in the dentate gyrus (DG) subgranular zone (SGZ) was determined using anti-doublecortin antibodies (anti-DCX) (1:500, ab18723; Abcam, Cambridge, MA, USA). Proliferative activity in the DG SGZ was determined by immunohistochemical staining of the nuclear antigen of proliferating cells (PCNA) using mouse monoclonal anti-PCNA antibodies (1:500, ab29) (Abcam, Cambridge, MA, USA). Secondary antibodies conjugated to horseradish peroxidase (PI-1000, anti-rabbit; PI-2000, anti-mouse) were used according to the manufacturer’s recommendations (1:100; Vector Laboratories, Burlingame, CA, USA).
7
Immunoblotting for GST-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PIP strip membranes (P-6001 – Echelon) were blocked with 0.1% ovalbumin (in TBST - 0.1M Tris, 1.5M NaCl, 0.1% Tween 20, pH 8.0) for two hours. The membranes were then incubated with 0.005 µg/ml of purified GST-tagged protein in 0.1% ovalbumin overnight at 4°C. After three washes with TBST for ten minutes each, the membranes were incubated with primary antibody for two hours. The following primary antibodies were used: 1:1500 mouse anti-Hts-1B1 and 1:10,000 mouse anti-GST (SAB4200237 – Sigma-Aldrich). The membranes were next washed twice with TBST and blocked twice with 0.1% ovalbumin for ten minutes each. The membranes were then incubated with a 1:2000 dilution of peroxidase-conjugated anti-mouse secondary antibody (PI-2000 – Vector Labs) for one hour, followed by four washes with TBST for 15 minutes each. Signal was detected using the BM Chemiluminescence Western Blotting Peroxidase Substrate. Using Photoshop, the film image was inverted and the signal intensity for each lipid spot was measured as mean gray value normalized against the blank. Experiments were done in triplicate with data expressed as absolute values to the control and presented as ‘mean±sem’. Student's t-tests were performed for all statistical comparisons.
8
Western Blot Analysis of β-III-Tubulin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A standard Western Blott protocol was used as previously described18 (link). hPDL-derived cells (in basal media) were harvested using trypsin/EDTA (Gibco), washed twice with PBS, resuspended in RIPA lysis buffer (Millipore) in the presence of protease inhibitors (PierceTM. protease inhibitor Mini Tables, Pierce Biotechnology Inc) and PMSF 1 M (Abcam) for 30 min at 4 °C. Protein concentration was determined using the bradford protein assay (Sigma-Aldrich). Proteins were separated in 8% SDS-polyacryamide gel (PAGE-SDS) and transferred to a nitrocellulose membrane (Whatman). PageRuler™ Prestained Protein Ladder (Thermo Scientific) has been used as size standards in protein electrophoresis (SDS-PAGE) and Western-Blotting. After transfer, nitrocellulose membranes were stained with Ponceau S solution (Sigma-Aldrich) to visualize protein bands. Blots were then incubated over-night at 4 °C with rabbit antibody against β-III-tubulin (TUJ1; 1:1000, Covance). Secondary antibody was used at 1:7000 for peroxidase anti-mouse Ab (PI-2000, Vector Laboratories). Immunoreactivity was detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Biosciences Europe) and LuminataTM Forte (Millipore corporation) using ImageQuant LAS 500 Gel Documentation System (GE Healthcare). The molecular weight of β-III-tubulin is approximately 55 kDa.
9
Immunohistochemical Analysis of Hippocampal Microglia, Neurogenesis, and Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to evaluate the activity of microglia/macrophages in the mouse hippocampi, immunostaining was performed using anti-Iba-1 rabbit polyclonal antibodies (1:500, ab108539) and anti-CD86 rabbit monoclonal antibodies (1:1,000, ab53004; both from Abcam, Cambridge, MA, USA). For the analysis of neurogenesis, the determination of the number of newly-formed neurons in the dentate gyrus (DG) subgranular zone (SGZ) was performed using anti-doublecortin (anti-DCX) antibody (1:500, ab18723; Abcam). Appropriate secondary antibodies conjugated to horseradish peroxidase (PI-1000, anti-rabbit; PI-2000, anti-mouse) were used according to the manufacturer's instructions (1:100; Vector Laboratories, Burlingame, CA, USA). Proliferating cell nuclear antigen (PCNA)-immunoreactivity was determined using anti-PCNA mouse monoclonal antibodies (1:500, ab29) and NeuN rabbit monoclonal antibodies (1:1,000, ab177487; both from Abcam) in the DG SGZ. Appropriate fluorescent secondary antibodies (anti-mouse, ab150108; anti-rabbit, ab150080) were used according to the manufacturer's instructions (1:500; Abcam). Following incubation with the antibodies (4°C, 24 h for primary antibodies and room temperature, 45 min for secondary antibodies), the sections were embedded in Fluoroshield mounting medium with DAPI (ab104139; Abcam).
10
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed as described [52 (link)] using the following antibodies and dilutions: 1:1000 Uhrf1 (sc-373750, Santa Cruz Biotechnology), 1:1000 Rb (9313T, Cell Signaling), actin (A1978, Sigma), E2F1 (3742S, Cell Signaling), E2F2 (sc-9967, Santa Cruz Biotechnology), E2F3 (MA5-11319, Nalgene Nunc); 1:1000 SEMA3E (PA547469, Thermo Scientific); 1:1000 AMPK (5831S, Cell Signaling); 1:1000 pAMPK (ab133448, Abcam). Secondary antibodies were diluted 1:1000 (PI-1000, PI-2000 or PI-9500, Vector Laboratories). Bands were visualized using chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific). Band intensities were analyzed using ImageJ software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!