To obtain total lysates for immunoblotting analyses, primary hippocampal neurons were washed in phosphate buffered saline (PBS) and collected by gentle scraping in ice-cold RIPA buffer containing (in mM) 50 Tris pH 7.4, 100 NaCl, 1 EGTA, 1 PMSF, 1 sodium orthovanadate, 1 NaF, 0.5% NP-40, and 0.2% SDS supplemented with protease inhibitor cocktail II (Roche Diagnostic, Monza, Italy). Nitrocellulose membranes were incubated overnight with rabbit-polyclonal anti-NaV1.6 (1:500, Alomone Labs-Israel)22 (link),80 (link), anti-NaV1.2 (1:500, Alomone Labs-Israel)22 (link) and anti-NaV1.1 antibodies (1:500, Alomone Labs-Israel)65 (link), or mouse monoclonal anti-tubulin (1:3000, Sigma Aldrich, Milan, Italy). Immunoreactive bands were detected with the chemiluminescence system (Amersham-Pharmacia-Biosciences, UK). Films were developed with a standard photographic procedure and the quantitative analysis of detected bands was carried out by densitometric scanning.
Anti nav1
Manufactured by Alomone
Sourced in Israel, United States
Anti-Nav1.6 is a research tool that targets the sodium channel protein Nav1.6. It can be used to study the function and regulation of this ion channel in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor mixture cocktail, by Roche (3 mentions)
Protease inhibitor cocktail 2, by Roche (2 mentions)
Anti tubulin, by Merck Group (2 mentions)
Anti ankg, by Santa Cruz Biotechnology (2 mentions)
Anti ha, by Abbkine (2 mentions)
Quinidine, by Maclin Biochemical Technology (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Np 40 lysis buffer, by Beyotime (2 mentions)
Anti flag, by Abbkine (2 mentions)
Anti cav1, by Alomone (2 mentions)
Anti flag, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti mt2, by Abcam (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Anti p p50, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti nav1, by Alomone (1 mentions)
Anti p65, by Santa Cruz Biotechnology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
14 protocols using anti nav1
1
Immunoblotting Analysis of Sodium Channels
2
Investigating Sodium Channel Interactome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Commercial antibodies used were anti-AnkG (Santa Cruz), anti-Slack (NeuroMab), anti-NaV1.2 (Alomone), anti-NaV1.6 (Alomone), anti-HA (Abbkine), anti-Flag (Abbkine), anti-β-actin (Biodragon), HRP goat anti-mouse IgG LCS (Abbkine), HRP mouse anti-rabbit IgG LCS (Abbkine), Alexa Fluor 488-AffinityPure Fab Fragment donkey anti-rabbit IgG (Jackson), and Alexa Fluor 594 donkey anti-mouse IgG (Yeason). GPCR Extraction Reagent was from Pierce, NP40 lysis buffer was from Beyotime, protease inhibitor mixture cocktail was from Roche Applied Science, rabbit IgG and mouse IgG were from Santa Cruz, and Protein G Dynabeads were from Invitrogen. Tetrodotoxin was from Absin Bioscience. Quinidine was from Macklin, and riluzole was from Meilunbio. All other reagents were purchased from Sigma-Aldrich.
3
Immunofluorescence Analysis of DRG Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ten days after DiI injection, control and NCI rats were perfused transcardially with 150 mL phosphate-buffered saline followed by 150 mL ice-cold 4% paraformaldehyde in PBS. DRGs (T13-L2) were removed and post fixed for 4 h in PFA and then gradiently dehydrate with 20% and 30% sucrose (24 h each, sink to the bottom as the sign of dehydration completion). 14 μm frozen sections contained DRGs (T13-L2) area were used in immunofluorescence study as described previously.32 (link)
The primary antibodies were anti-MT2 (1:200; Abcam, Cambridge, England), anti-Nav1.8 (1:200, Alomone Labs, Israel). The secondary antibodies were Alexa Fluor 488 (1:100, Life Technologies Inc.) and Alexa Fluor 555 (1:500, Life Technologies Inc.). Images were captured with AXIO SCOPE A1 (20X, ZEISS, Oberkochen, Germany).
The primary antibodies were anti-MT2 (1:200; Abcam, Cambridge, England), anti-Nav1.8 (1:200, Alomone Labs, Israel). The secondary antibodies were Alexa Fluor 488 (1:100, Life Technologies Inc.) and Alexa Fluor 555 (1:500, Life Technologies Inc.). Images were captured with AXIO SCOPE A1 (20X, ZEISS, Oberkochen, Germany).
4
Immunoblotting Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were separated by 10% SDS-PAGE and electroblotted onto PVDF membranes (Roche). After blocking, the blots were then incubated with monoclonal or polyclonal primary antibodies (mAb or pAb), followed by secondary IRDye® 680RD goat anti-mouse (1:5000) or goat anti-rabbit (1:5000) antibody (LI-COR Biosciences, Lincoln, NE, USA). Rabbit anti- Nav1.7 (1:500) and anti-Nav1.8 (1:500) pAbs were purchased from Alomone Labs (Jerusalem, Israel); mouse anti-p50 (1:200), anti-p65 (1:200) and anti-p-p50 (1:500) mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit anti-p-p65 (1:500) mAb were purchased from Cell Signaling Technology (Danvers, MA, USA); mouse anti-ADRB2/β2AR (1:1000) mAb and rabbit anti-p-ADRB2/β2AR (1:500) pAb were purchased from Proteintech (Wuhan, Hubei, China) and Sigma-Aldrich (USA), respectively; mouse anti-GAPDH (1:1000) and anti-SMN (1:1000) mAbs were purchased from MultiSciences (Hangzhou, Zhejiang, China) and BD Biosciences (San Diego, CA, USA), respectively. Protein signals were analyzed with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA).
5
Quantifying NaV1.7 and NaV1.8 in Diabetic Rats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expressions of NaV1.7 and NaV1.8 in colonic DRGs (T13-L2 DRGs) from diabetic and CON rats were determined using western blotting analysis, as previously described in detail.15 The primary antibodies used to probe the target proteins included rabbit anti-NaV1.7 or anti-NaV1.8 (1:200, Alomone Labs, Jerusalem, Israel), rabbit anti-GAPDH (1:1000, Biotechnology Co., CHN), and mouse anti-actin (1:1000; Chemicon, Temecula, CA, USA). The secondary antibodies included anti-rabbit peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-mouse horseradish peroxidase-conjugated secondary antibody (1:4000; Chemicon).
6
Ion Channel Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HL-1 cells were lysed using RIPA buffer (Thermo Fisher, Waltham, MA) containing protease inhibitor cocktail (Roche, South San Francisco, CA). Equal amounts of protein (10–20 μg) were loaded into wells of 7% or 3–8% tris-acetate gels, subjected to SDS-PAGE, and transferred to a 0.45 μm PVDF membrane (Thermo Fisher). Western blot analyses were performed using the following primary antibodies: anti-Flag (Sigma-Aldrich), anti-Kv4.3 (Alomone, Jerusalem, Israel), anti-Kv11.1 (Alomone), anti-Nav1.5 (Alomone), anti-Cav1.2 (Alomone), and anti-GAPDH (Millipore, Billerica, MA). The immunodensity of bands corresponding to targeted proteins were quantified using NIH ImageJ software, and values were corrected for protein loading by dividing them by GAPDH immunodensity signals in the same lanes. Subsequently, corrected values obtained from Western blots loaded with protein lysate from BKα-transfected cells were normalized to corrected values obtained from Null-transfected cells corresponding to the same ion channel. According to this analysis, by definition the average normalized intensity of Null transfection is unity. For Western blots designed to detect Kv11.1, both bands of the Kv11.1 doublet revealed by anti-Kv11.1 blotting were included in calculations.
7
Protein Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins of the cells were lysed in a RIPA buffer containing 1% protease inhibitors (Beyotime, Shanghai, China) and collected. After SDS-PAGE, the proteins were transferred onto PVDF membranes. The primary antibodies were anti-Cav1.2 (Alomone, Jerusalem, Israel), anti-Nav1.5 (Alomone, Jerusalem, Israel), anti-Kv4.2 (Abcam, Cambridge, England), anti-HOXa10 (ABclonal, Wuhan, China), and anti-β-Actin (ABclonal, Wuhan, China). The HRP-conjugated secondary antibody HRP-labeled goat anti-rabbit IgG (Absin, Shanghai, China) was used.
8
Quantitative Analysis of Myelination Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals were euthanized and perfused with PBS and 4% (w/v) paraformaldehyde (158127, Sigma). Coronal sections (16 μm) of brains blocked by 5% (v/v) goat serum were incubated with primary antibodies and corresponding secondary antibodies. Antibodies used were as follows: anti-MBP (sc271524, Santa Cruz), anti-NF200 (N0142, Sigma), anti-β-APP (51-2700, Invitrogen), anti-Caspr (MABN69, Millipore), anti-Nav1.6 (ASC-009, Alomone, Jerusalem), anti-PDGFRα (3174, Cell Signaling Technology), anti-Olig2 (ab9610, Millipore), Alexa Fluor 594 goat-anti-rabbit IgG (A-11012, Invitrogen), Alexa Fluor 594 goat-anti-mouse IgG (A-11005, Invitrogen), Alexa Fluor 488 goat-anti-rabbit IgG (A-11008, Invitrogen) and Alexa Fluor 488 goat-anti-mouse IgG (A-11001, Invitrogen). Images were obtained by fluorescence microscope (Nikon ECLIPSE Ci) or confocal microscope (TCS SP8 STED, Leica). At least three microscopic fields around the lesion were captured in each section. Three sections, at the sites of anterior, middle and posterior of the hematoma, were chosen for each mouse. Data were analyzed by an investigator who was blinded to the experimental setup.
9
Comprehensive Antibody Panel for Epigenetic Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Commercial antibodies used were: anti-FLAG (Sigma), anti-CDYL (Sigma, HPA035578), anti-REST (Santa Cruz, sc-25398), anti-EZH2 (Proteintech, 21800-1-AP), anti-HDAC1 (sc-7872), anti-HDAC2 (sc-7899), anti-SIRT1 (sc-15404), anti-FOS (ABclonal, A0236), anti-SET8 (CST, #2996), anti-UTX (sc-79334), anti-KDM5B (sc-67035), anti-CtBP (Proteintech, 10972-1-AP), anti-MeCP2 (ABclonal, A5694), anti-Nav1.6 (Alomone, ASC-009), anti-H3K9me2 (Abcam, ab1220), anti-H3K9me3 (Abcam, ab8898), anti-G9a (Millipore, 09-071), anti-H3K27me3 (Millipore, 07-449), anti-H3K27ac (Abcam, ab4729), anti-H3K4me1 (Abcam, ab8895), anti-H3K4me3 (Abcam, ab8580), anti-GAPDH and anti-Actin (MBL). A CDYL antibody that we generated (peptides antigen: DGFQGESPEKLDPVDQG and IDDRRDQPFDKRLRFSV; B&M) was used for ChIP-seq, western blotting with rat/mouse cell lysates, and immunohistochemistry. Protein A/G beads were from GE Healthcare Biosciences, protease inhibitor mixture cocktail was from Roche Applied Science. Tetrodotoxin, bicuculline, CGP 55845, and DL-AP5 were from Abcam. Neurobiotin was from Vector Laboratories. Streptavidin Alexa Fluor 488 was from Life Technologies. 4,9-TTX was from Focus Biomolecules. All other reagents were purchased from Sigma-Aldrich.
10
Quantification of Voltage-Gated Sodium Channels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole brain membrane proteins were isolated from WT and Scn2aE/+ mice at 4 weeks of age. Membrane fractions (50 μg/lane) were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. Blots were probed with anti-NaV1.2 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) or anti-Nav1.1 (Alomone Labs, Jerusalem, ISR; #ASC-001, RRID: AB_2040003; 1:200 dilution) or anti-Nav1.6 (Alomone Labs, Jerusalem, ISR; #ASC-009, RRID: AB_2040202; 1:500 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard. Alexa Fluor 790 goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA; #111-655-144, RRID: AB_2338086; 1:10 000 dilution) and 680 goat anti-mouse antibody (Jackson ImmunoResearch; #115-625-146; RRID: AB_2338935; 1:10 000 dilution) were used to detect signal on an Odyssey imaging system (Li-COR, Lincoln, Nebraska USA). Relative protein levels were determined by densitometry with Image Studio (Li-COR) and expressed as a ratio of NaV1.x to GRP75 (normalized to the mean ratio for WT mice), with 7–8 biological replicates per genotype, balanced by sex. Statistical comparison between groups was made using Student’s t-test (GraphPad Prism).
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!