For western blot analysis, cultured neurons were lysed in RIPA buffer (Thermo Scientific) containing proteinase and phosphatase inhibitors (Roche Diagnostics). Equal amounts of protein were separated on 4–20 % SDS PAGE gradient gels (Bio-Rad) and then transferred to PVDF or nitrocellulose membranes (Bio-Rad) for immunodetection. Primary antibodies used were: ATM2c1 and 53BP1 (Abcam); Actin (Santa Cruz Biotechnology); GFAP, GS, mTOR and P-mTORs2448 (Cell Signaling); and tau3R (Millipore). Secondary antibodies were chicken anti-rabbit IgG-HRP and chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology). Chemiluminescent substrates used were SuperSignal™ West Pico Chemiluminescent Substrate and SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific). For immunofluorescence staining, neurons were fixed in 4 % paraformaldehyde for 30 min. Fixed cells were then incubated in blocking buffer (10 % goat serum, 0.5 % Triton X100 in PBS) for 1 h. Primary antibody HDAC4 (1:1000, Abcam) incubation were carried out in 4°, overnight. Alexa linked secondary antibodies (Life Technologies) were used to detect the presence of the HDAC4 antigens. Stained cells were photographed and viewed at a final magnification of 200 using Leica Application Suite/Leica DM5000B.
Nitrocellulose or pvdf membranes
Manufactured by Bio-Rad
Sourced in United States
Nitrocellulose or PVDF membranes are widely used as solid supports in various biochemical and immunological techniques, such as Western blotting, dot blotting, and protein-based array analysis. These membranes serve as a platform for the immobilization and detection of proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Semidry transfer system, by Thermo Fisher Scientific (4 mentions)
Chemiluminescence kit, by PerkinElmer (3 mentions)
Anti d1r antibody, by Merck Group (2 mentions)
Anti d2r antibody, by Alomone (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Proteinase and phosphatase inhibitor, by Roche (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Gradient sds page gel, by Bio-Rad (1 mentions)
Chicken anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Application suite dm5000b, by Leica (1 mentions)
Chicken anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Hdac4, by Abcam (1 mentions)
Peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Supersignal westfemto ecl reagent, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Mouse monoclonal anti β actin c4, by Santa Cruz Biotechnology (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
11 protocols using nitrocellulose or pvdf membranes
1
Neuronal Protein Analysis: Western Blot and Immunofluorescence
2
Integrin Immunoblotting and Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were resolved by SDS-PAGE under non-reducing conditions, blotted onto PVDF or nitrocellulose membranes (Bio-Rad, Hercules, California, USA), and probed with rabbit monoclonal antibodies against integrins αv, αvβ3, αvβ5, αvβ6, or αvβ8 ([27] (link), kindly provided by Merck KGaA, Darmstadt, Germany). The antigens were detected using a peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and SuperSignal WestFemto ECL reagent (Thermo Fisher Scientific). Chemiluminescence signals were captured with a GEL LOGIC 2200 imaging system and analysed with the Kodak MI SE software. For the detection of the αvβ1 complex immunoprecipitation was performed with a β1 antibody (Millipore clone P4C10, Merck KGaA). The immune complexes were eluted in non-reducing sample buffer, resolved by SDS-PAGE, immunoblotted and detected with the rabbit monoclonal antibody against αv.
3
Western Blot Analysis of Spliceosomal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were resolved by SDS-PAGE and transferred to PVDF or nitrocellulose membranes (BioRad). Membranes were blocked and then incubated with the primary antibody. After washing, membranes were incubated with HRP-conjugated (BioRad) or IRDye® 800CW (LI-COR Biosciences) secondary antibodies. Bound antibody was detected using ECL plus reagent (GE Healthcare) or an Odyssey imaging system (LI-COR Biosciences). Western blots were performed at least three times, and representative images are shown in each case. The antibodies used were mouse monoclonal anti-βactin C4 (Santa Cruz Biotechnology), mouse monoclonal anti-SUMO2 8A2, mouse monoclonal anti-T7 tag (Novagen), rabbit monoclonal anti Prp3 (St John's Laboratory), rabbit polyclonal anti-60K/Prp4 (45 (link)), rabbit polyclonal anti-SF3a120 (46 (link)) and rabbit polyclonal anti-Snu114 (47 (link)).
4
Quantifying ABCC4 and ABCG2 Proteins in HT-29 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins isolated from HT-29 cells were extracted with NP-40 lysis buffer (50 mM Tris, pH 8.0, containing 1% Nonidet-Igepal, 150 mM NaCl, 5 mM EDTA) with the Halt protease inhibitor cocktail (Thermo Scientific, Waltham, MA, USA), and the soluble protein fraction was collected through centrifugation. The protein concentrations in the cell lysates were measured with the BCA method (Pierce/Thermo Scientific, Waltham, MA, USA) and were equalized between samples. The protein extracts were subjected to SDS-PAGE analysis and were electro transferred onto PVDF or nitrocellulose membranes (BioRad, Hercules, CA, USA) followed by immunodetection goat anti human ABCC4 #PA5-18315 (Thermo Fisher Scientific), rabbit anty human ABCG2 #ORB155559 (Biorbyt). The control-mouse rabbit anti-α-tubulin antibody conjugated with HRP (NB100-690H) was obtained from Novus Biologicals (Centennial, CO, USA) and used as a loading control. Detection was performed using secondary HRP-conjugated antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) followed by incubation with an enhanced chemiluminescence kit (Thermo Scientific, Waltham, MA, USA) and development with Kodak BioMax Light Film (Eastman Kodak, Rochester, NY, USA).
5
Co-immunoprecipitation of D1R and D2R in Rat Brain
6
Choroidal Protein Quantification Post-Burn
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assessment of choroidal protein levels, eyes were enucleated from mice 3 days after burn. RIPA buffer with anti-protease and anti-phosphatase (BioRad) was freshly prepared to manually with a piston to homogenize tissues and for cells lysis. Protein concentration was assessed by BCA assay (Sigma-Aldrich, Oakville, CA), and 30μg of protein analyzed for each condition by standard SDS-PAGE technique using Bis-Acrylamide gel 10% or 12.5% depending of protein size. Total protein transfer on nitrocellulose or PVDF membranes (Bio-Rad, Mississauga, ON, CA) was evaluated with Ponceau Red (Sigma-Aldrich, Oakville, CA). Antibody solutions and dilutions were prepared as per manufacturers’ recommendations.
7
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with cold PBS and lysed in RIPA buffer (#R0278, Sigma-Aldrich) containing protease and phosphatase inhibitor cocktails (Roche). The total protein concentration in the lysates was determined using the BCA kit (#BCA1, Sigma-Aldrich). The proteins were mixed with 5X SDS-PAGE buffer (Biosesang), separated using SDS-PAGE (8% or 12%), and then transferred to nitrocellulose or PVDF membranes (Bio-Rad, Hercules, CA, USA) at 70 V for 60 min. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing Tween-20 (TBST) for 1 h at 4 °C to prevent nonspecific binding. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies diluted at 1:500 or 1:1000. After washing with TBST, the membranes were incubated with the appropriate secondary antibodies (Sigma-Aldrich) for 1 h at 25 °C. The immunoblots were developed using a chemiluminescent substrate (Thermo Fisher Scientific, Sunnyvale, CA, USA). Protein expression was then quantified using ImageJ (version 1.54d, NIH, Bethesda, MD, USA).
8
Western Blot Analysis of Synaptic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue corresponding to the NAc or CPu was collected from brains of each male or female rat (N = 6–9) rapidly after sacrifice. Following homogenization, 30–50 μg of protein from the indicated region were incubated in sample buffer for 3–5 min at 95 °C. Proteins were resolved by electrophoresis on 10% polyacrylamide gels under denaturing conditions (SDS–PAGE) and transferred onto nitrocellulose or PVDF membranes (Bio-Rad Laboratories, Hercules, CA) using a semidry transfer system (Invitrogen). Membranes were blocked in TBS-Tween (TBS-T)/5% nonfat milk for 1 h followed by incubation with PBS-T/5% nonfat milk containing the indicated first antibody overnight at 4 °C. Membranes were washed in TBS-T (3 × 10 min) and incubated with the appropriate horseradish peroxidase (HRP)-conjugated polyclonal secondary antibody (Bio-Rad) for 2 h at room temperature. After three washes as indicated above, signal detection was performed using a chemiluminescence kit (Perkin-Elmer). The primary antibodies used were anti-phosphoGSK-3 (1:1000, Cell Signaling), anti-BDNF (1:1000, Abcam), anti-phosphoTrkB (1:1000, Cell Signaling), and rabbit anti-GAPDH (1:10,000 or 1:20,000 Abcam).
9
Western Blot Analysis of Sez6 Family Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentrations were quantified using the DC Protein Assay kit (Bio-Rad, USA) and 40 µg of total protein were used for Western Blot analysis. Samples were boiled for 5 min at 95 °C in Laemmli buffer. For the detection of Sez6L, Laemmli buffer without the disulfide bridge reducing agent β-mercaptoethanol was used and samples were not boiled13 (link). Samples were separated on 7.5% Mini-PROTEAN TGX Stain-Free gels (Bio-Rad). Nitrocellulose or PVDF membranes (Bio-Rad) were incubated with primary antibody overnight at 4 °C. After incubation with secondary antibody at room temperature for 1 h, membranes were developed with Clarity ECL (Bio-Rad). The following antibodies were used: monoclonal Sez6 (1:100; see Ref.13 (link)), monoclonal Sez6L (1:100; Ref.13 (link)), polyclonal Sez6L2 (1:500; R&D Systems, AF4916), β actin (1: 1000; Sigma, A5441) and monoclonal APP (Y188; 1:6000; Abcam, ab32136), HRP coupled anti-rat, anti-rabbit, anti-goat and anti-mouse (Life Technologies). Western blot protein levels were normalized to total blot protein using BioRad stain-free technology and the ChemiDoc imager as described23 (link).
10
Co-immunoprecipitation of Dopamine Receptors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Co-immunoprecipitation was performed as previously described [14 (link), 19 (link)]. Protein homogenates (250–300 μg) from rat NAc or CPu were incubated with an anti-D2R antibody (Alomone Laboratories) at 4 °C overnight under gentle rotation. After adding 40–50 μl of protein G/A, the mixture was further incubated for 1 h. After 3 washes with PBS-Tween, SDS buffer (70 μl) was added, and the immunoprecipitates were incubated for 5 min at 95 °C. Proteins were resolved by electrophoresis on 10% polyacrylamide gels under denaturing conditions (SDS-PAGE) and transferred onto nitrocellulose or PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) using a semidry transfer system (Invitrogen, Carlsbad, CA, USA). Membranes were incubated in PBS-Tween (PBS-T)/10% nonfat milk for 1 h. After 3 washes, membranes were incubated with PBS-T/5% nonfat milk containing the anti-D1R antibody raised in rats (Sigma, St. Louis, MO, USA). Membranes were washed once in PBS-T and 2 times in PBS (10 min each) and incubated with the appropriate horseradish peroxidase (HRP)-conjugated polyclonal secondary antibody for 2 h. After 3 washes as indicated above, signal detection was performed using a chemiluminescence kit (Perkin-Elmer).
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!