Nitrocellulose blotting membrane

Manufactured by Bio-Rad

Sourced in United States

Nitrocellulose blotting membranes are a type of laboratory equipment used for the detection and analysis of proteins in Western blot assays. They provide a surface for the immobilization of proteins, allowing for their subsequent detection and quantification using specific antibodies.

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Lab products found in correlation

Ripa buffer, by Thermo Fisher Scientific (2 mentions) Mini protean tgx gel, by Bio-Rad (2 mentions) Odyssey system, by LI COR (2 mentions) Enhanced chemiluminescence, by Bio-Rad (2 mentions) Immobilon western, by Merck Group (2 mentions) Enhanced chemiluminescence detection kit, by Bio-Rad (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) β actin, by Merck Group (1 mentions) Protease inhibitor mixture, by Roche (1 mentions) Ab108341, by Abcam (1 mentions) Anti β actin, by Cell Signaling Technology (1 mentions) Pierce rapid gold bca protein assay, by Thermo Fisher Scientific (1 mentions) Ab32072, by Abcam (1 mentions) Flag tag m2, by Merck Group (1 mentions) Laemmli sample buffer, by Bio-Rad (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Tim23, by BD (1 mentions) Micu1, by Abcam (1 mentions) Cytochrome c, by Cell Signaling Technology (1 mentions) Vdac1, by Abcam (1 mentions)

Close spelling variants of this product

Nitrocellulose membranes (5040 citations) Nitrocellulose (194 citations)

17 protocols using nitrocellulose blotting membrane

Isolation and Analysis of Mitochondria from Dentate Gyrus

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Percoll density gradient centrifugation was used to isolate the mitochondria. Tissues in the DG region were dissected out as described previously (Hagihara et al. 2009 ) and homogenized in isolation buffer (225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM HEPES, pH = 7.2). After centrifugation at 1,100 g for 2 min, the supernatant was mixed with 80% Percoll and carefully layered on the top of 10% Percoll, then centrifuged at 18,500 g for 10 min. The myelin-rich top fraction was removed, leaving the mitochondrion-enriched pellet at the bottom. The isolated mitochondria were washed once and then lysed with SDS sample buffer.
A sample containing 10 to 20 μg of total protein was loaded onto 8% or 14% sodium dodecylsulfate (SDS) gels. Following electrophoresis, gels were transferred onto Nitrocellulose Blotting Membranes (Bio-Rad). Antigen-specific primary antibodies, including rabbit anti-Drp1 (1:200; Cell Signaling, RRID:AB_11178938), rabbit anti-phospho-Drp1 (1:1000, Cell Signaling, RRID:AB_11178659), mouse anti-Mfn2 (1:2000; Abcam, RRID:AB_2142629), mouse anti-beta-Catenin (1:2000; Abcam, RRID:AB_562065), rabbit anti-Vps35 (1:200; Abcam, RRID:AB_1524565), mouse anti-VCP (1:2000; Millipore, RRID:AB_10806328), mouse anti-Atp1a1 (1:1000; Santa Cruz, RRID:AB_1125502), mouse anti-tubulin (1:8000; Proteintech, RRID:AB_2687491), and rabbit anti-VDAC (1:5000, Abcam, RRID:AB_297264).

Tang F.L., Wang J., Itokazu Y, & Yu R.K. (2020). Ganglioside GD3 regulates dendritic growth in newborn neurons in adult mouse hippocampus via modulation of mitochondrial dynamics. Journal of neurochemistry, 156(6), 819-833.

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Protein Extraction and Western Blotting

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The cells were lysed in RIPA buffer (Thermo Fisher Scientific) and centrifuged at 14,000 rpm at 4 °C for 10 min. Protein concentration was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on 10 or 4–20% gradient Mini-PROTEAN TGX gels (Bio-Rad) and transferred to nitrocellulose blotting membranes (Bio-Rad). Western blot and immunoreactive proteins were developed using an enhanced chemiluminescence detection kit (Bio-Rad).

Martín-Maestro P., Sproul A., Martinez H., Paquet D., Gerges M., Noggle S, & Starkov A.A. (2019). Autophagy induction by Bexarotene promotes mitophagy in Presenilin 1 familial Alzheimer’s disease iPSC-derived neural stem cells. Molecular neurobiology, 56(12), 8220-8236.

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Modulation of COX-1 and COX-2 Expression

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HUVECs were treated with indomethacin, HRF, and the extracts (1, 10, and 100 µg/mL) prior to IL-1β (1 ηg/mL) stimulation for 24 h. The COX determination, including a Bradford protein assay, was performed as previously described [21 (link)]. Briefly, all samples were loaded into SDS-PAGE, underwent electrophoresis, and were transferred to nitrocellulose blotting membranes (Bio-Rad, Germany). After blocking with a solution of 5% skim milk for 1.5 h at room temperature, the membranes were incubated overnight with a specific monoclonal COX-1 or COX-2 antibody at 4°C and an anti-mouse IgG of COX-1 or anti-COX-2 (Sigma-Aldrich, USA, dilution 1/10000) for 1.5 h, respectively. β-Actin (Sigma-Aldrich, USA, dilution 1/5000) was used as an internal control in the experiment. The COX protein bands were visualized using VersaDoc™ Imaging Systems (Bio-Rad, Germany).

Palo T., Thaworn A., Charoenkij P., Thamsermsang O., Chotewuttakorn S., Tripatara P., Laohapand T, & Akarasereenont P. (2017). The Effects of Thai Herbal Ha-Rak Formula on COX Isoform Expression in Human Umbilical Vein Endothelial Cells Induced by IL-1β. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 9383272.

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Western Blot Analysis of Protein Samples

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After harvesting, the cells were lysed in ice-cold lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1% SDS, 1× protease inhibitor mixture (Roche, Basel, Switzerland)) for 30 min and centrifuged at 4 °C, 13,000 rpm for 10 min to remove insoluble material. The soluble protein concentration was determined by Bradford assay. Protein samples (60 μg) were separated by SDS-PAGE and transferred to nitrocellulose blotting membranes (Bio-Rad, Hercules, CA, USA). The membranes were treated with block buffer (5% non-fat milk in 0.1% TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20)) at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4 °C, then washing (3 × 12 min) in PBS/Tween 20, followed by incubating with secondary antibodies in blocking buffer at room temperature for 2 h. Finally, washing (3 × 12 min) in PBS/Tween 20 again. The signals were detected and measured using LICOR Odyssey system (LI-COR, Lincoln, NE, USA). All the western blots were repeated at least three times.

Wang Q., Zhang X., Chen L., Weng S., Xia Y., Ye Y., Li K., Liao Z., Chen P., Alsamman K., Meng C., Stevens C., Hupp T.R, & Lin Y. (2019). Regulation of the Expression of DAPK1 by SUMO Pathway. Biomolecules, 9(4), 151.

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Western Blot Analysis of Murine VP Tissue

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Fresh-frozen VP tissues from 12-week-old male FVB mice were sliced on ice with stainless steel disposable scalpels (Fisher Scientific) then homogenized in RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% TRITON-X) supplemented with phosphatases and protease inhibitors (Mini, Pierce™, Thermo Fisher) using a tissue grinder kit (Kontes). Equal amounts of protein (15 μg; Pierce™ Rapid Gold BCA Protein Assay, Thermo Fisher) were resolved on 8–12% Tris-glycine SDS-polyacrylamide gels and transferred to nitrocellulose blotting membranes (Bio-Rad), following standard procedures. Membranes were probed with the following antibodies according to the manufacturer’s instructions: rabbit monoclonal [Y69] anti-c-MYC (#ab32072, Abcam; dilution 1:1,000), rabbit monoclonal [ER179(2)] anti-AR (#ab108341, Abcam; dilution 1:1,000) or rabbit polyclonal anti-β-Actin (#4967, Cell Signaling Technology; dilution 1:1,000). Densitometry analyses were made with ImageJ (U.S. NIH, Bethesda, MD; http://imagej.nih.gov/ij/). Results were normalized to β-actin and expressed as arbitrary units.

Qiu X., Boufaied N., Hallal T., Feit A., de Polo A., Luoma A.M., Alahmadi W., Larocque J., Zadra G., Xie Y., Gu S., Tang Q., Zhang Y., Syamala S., Seo J.H., Bell C., O’Connor E., Liu Y., Schaeffer E.M., Jeffrey Karnes R., Weinmann S., Davicioni E., Morrissey C., Cejas P., Ellis L., Loda M., Wucherpfennig K.W., Pomerantz M.M., Spratt D.E., Corey E., Freedman M.L., Shirley Liu X., Brown M., Long H.W, & Labbé D.P. (2022). MYC drives aggressive prostate cancer by disrupting transcriptional pause release at androgen receptor targets. Nature Communications, 13, 2559.

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Mitochondrial Protein Immunoblotting

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Samples were lysed in RIPA buffer (Thermo Fisher Scientific) and centrifuged at 14,000 rpm at 4 °C for 20 min. Supernatants were diluted in 2X Laemmli sample buffer (Bio-Rad). Proteins were separated on 10 or 4–20% gradient Mini-PROTEAN TGX gels (Bio-Rad) and transferred to nitrocellulose blotting membranes (Bio-Rad). Membranes were probed with primary antibodies against FLAG tag (M2, Sigma), MCU (Sigma), MICU1 (Abcam, Cambridge, MA), cytochrome C (Cell Signaling, Danvers, MA), Cyclophilin D (Millipore, Billerica, MA), Tim23 (BD Biosciences, San Jose, CA), VDAC1 (Abcam), COX1 (Abcam), GAPDH (Thermo Fisher Scientific), citrate synthase (Abcam), and an oxidative phosphorylation (OXPHOS) cocktail (Abcam) overnight at 4 °C. Blots were then probed with horseradish peroxidase-conjugated anti-mouse (Jackson ImmunoResearch, West Grove, PA) or anti-rabbit (Thermo Fisher Scientific) secondary antibodies and detected using enhanced chemiluminescence (Bio-Rad).

Kawamata H., Peixoto P., Konrad C., Palomo G., Bredvik K., Gerges M., Valsecchi F., Petrucelli L., Ravits J.M., Starkov A, & Manfredi G. (2017). Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo. Molecular Neurodegeneration, 12, 37.

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Western Blot Analysis of CCRL2 Protein

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The cells were lysed in lysis buffer for 30 mins and centrifuged at 4°C, 13,000 rpm for 10 mins to remove insoluble material. The soluble protein concentration was determined by Bradford assay. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose blotting membranes (Bio-Rad, CA, USA). The membranes were treated with block buffer at room temperature for 1 hr. The membranes were then incubated with primary antibodies overnight at 4°C, then washing in PBS/Tween-20, followed by incubating with secondary antibodies in blocking buffer at room temperature for 2 hrs. Finally, washing in PBS/Tween-20 again. The signals were detected and measured using LICOR Odyssey system (LI-COR, NE, USA). Anti-GAPDH antibody (60004-1-Ig) and anti-CCRL2 antibody (66611-1-Ig) were purchased from Proteintech (Wuhan, China).

Li D., Zhong J., Zhang G., Lin L, & Liu Z. (2019). Oncogenic Role and Prognostic Value of MicroRNA-937-3p in Patients with Breast Cancer. OncoTargets and therapy, 12, 11045-11056.

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Western Blot Analysis of SCG Neuron Lysates

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Cellular lysates of SCG neurons, treated with media only or 1 × 10⁸ A1EVs/mL, were generated using radioimmunoprecipitation assay buffer (RIPA) lysis buffer (Thermo) plus the HALT protease inhibitor cocktail (Thermo) and prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) separation. Protein quantification of the lysates was performed using a BCA protein assay kit (Thermo Scientific). A total amount of 30 μg of proteins per well was separated on 4–12% SDS–polyacrylamide gels in quadruplicate and transferred onto nitrocellulose blotting membranes (Bio-Rad). Blocking of the membranes was performed using 5% non-fat milk in PBS containing 0.5% Tween 20. Neuron lysate blots were probed using the following primary antibodies: RUNX3 (1:1000 Fisher Scientific), TOM70 (1:100, Sigma), TrkA (1:1000, Novus Biologicals), ERK5 (1:1000, Novus Biologicals), p-ERK5 (1:1000, Fisher Scientific), CREB (1:1000, Novus Biologica), p-CREB (1:1000, Novus Biologica), β-actin (1:5000, Thermo Fishier Scientific). Following washing with TBS-Tween buffer and incubation with appropriate HRP-labelled secondary antibodies, protein detections were performed using ECL kit (Thermo Scientific). Blots were imaged using the Gel Doc XR+ system (BioRad) and analyzed using ImageJ (NIH).

Middleton R.C., Liao K., Liu W., de Couto G., Garcia N., Antes T., Wang Y., Wu D., Li X., Tourtellotte W.G, & Marbán E. (2023). Newt A1 cell-derived extracellular vesicles promote mammalian nerve growth. Scientific Reports, 13, 11829.

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Investigating cAMP Signaling in DRG Neurons

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The primary DRG neurons were treated with 20 μM PGE2 (14010, Cayman Chemical, Ann Arbor, MI) for 30 min; 10 μM PKA inhibitor (H-89, BML-E1196, Enzo Life Sciences, Farmingdale, NY) for 1 h. Western blot analysis was conducted on the protein lysates from the cultured primary DRG neurons. The supernatants of lysates were collected after centrifugation and separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then blotted on the nitrocellulose blotting membranes (Bio-Rad Laboratories). Specific antibodies were applied for incubation, and the proteins were detected by using an enhanced chemiluminescence kit (Amersham Bioscience, RNP2108). The antibodies used for western blot were p-CREB (1:1000, 9198, Cell Signaling Technology), CREB (1:000, 9197, Cell Signaling Technology), p-PKA (1:1000, 5661, Cell Signaling Technology), PKA (1:1000, 4782 S, Cell Signaling Technology), and β-tubulin (1:2000, MA5-16308, Invitrogen). The original blots are provided in the Source Data file.

Ni S., Ling Z., Wang X., Cao Y., Wu T., Deng R., Crane J.L., Skolasky R., Demehri S., Zhen G., Jain A., Wu P., Pan D., Hu B., Lyu X., Li Y., Chen H., Qi H., Guan Y., Dong X., Wan M., Zou X., Lu H., Hu J, & Cao X. (2019). Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice. Nature Communications, 10, 5643.

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Protein Expression Analysis by Western Blot

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Cells were seeded at 3×10⁵ cells per well in 6-well plates and duplicate wells were treated with siRNA at the indicated concentration for 48 hours. Cell lysates were prepared and total protein concentration determined by the BCA protein assay (ThermoFisher, Waltham MA). 20 µg aliquots of each sample were separated on 5–12% SDS-polyacrylamide gels, transferred to nitrocellulose blotting membrane (Bio-rad, Hercules CA) and immunoblotted with antibodies directed against ATM, ATIC (Abnova, Taiwan), NBN (Cell Signaling, Danvers MA), MTPAP (EMD Millipore, Billeria MA), p53 (Santa Cruz, Dallas TX), p21(Cip1 /WAF1), cyclin B, tubulin, AMPK or Phospho-T172 AMPK (Cell Signaling, Danvers MA), all at a dilution of 1:1000, and incubated at 4 °C overnight. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature, washed and developed using enhanced chemiluminescence protocols (ECL; GE Health Care Life Science, Piscataway Township NJ) and quantified using ImageQuant software (GE Health Care Life Science, Piscataway Township NJ).

Liu X., Paila U.D., Teraoka S.N., Wright J.A., Huang X., Quinlan A.R., Gatti R.A, & Concannon P. (2017). Identification of ATIC as a novel target for chemoradiosensitization. International journal of radiation oncology, biology, physics, 100(1), 162-173.

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