Startingblock pbs blocking buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

StartingBlock PBS blocking buffer is a ready-to-use solution designed to block non-specific binding sites in immunoassays and Western blotting applications. It is a phosphate-buffered saline (PBS) formulation that effectively minimizes background signals, thereby improving the specificity and sensitivity of protein detection.

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Lab products found in correlation

Acrylamide gel, by Bio-Rad (2 mentions) Pro q emerald 300, by Thermo Fisher Scientific (2 mentions) Immobilon p transfer membrane, by Merck Group (2 mentions) Sypro ruby, by Thermo Fisher Scientific (2 mentions) Chemidoc mp imaging system, by Bio-Rad (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Supersignal west pico, by Thermo Fisher Scientific (1 mentions) Tgx stain free sds page gel, by Bio-Rad (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Hrp conjugated secondary ab, by Cell Signaling Technology (1 mentions) Ripa buffer, by Nacalai Tesque (1 mentions) Protease inhibitor cocktail, by Nacalai Tesque (1 mentions) Chemi lumi one super, by Nacalai Tesque (1 mentions) Anti β actin ab, by Cell Signaling Technology (1 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Caspase 3 d3r6y, by Cell Signaling Technology (1 mentions) Bax d2e11, by Cell Signaling Technology (1 mentions)

10 protocols using startingblock pbs blocking buffer

Retinal Protein Immunoblotting Assay

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Following euthanasia eyes were removed, and the retina was dissected from the eye and snap frozen on dry ice. Single retinas (n = 3 per genotype) were homogenized independently in RIPA buffer (150 mM NaCl, 50 nM Tris, pH 8.0, 1% IGEPAL, 0.5% Deoxycholate, 0.1% SDS) containing 1X protease inhibitors (Roche). Lysate concentration was determined using a Pierce BCA protein assay kit (Thermo) and samples were equally loaded and separated on a 4–15% TGX Stain-Free SDS-PAGE gel (Bio-Rad). Following electrophoresis, separated proteins were wet transferred to PVDF for immunoblotting. Blots were incubated in StartingBlock PBS blocking buffer (Thermo) for >1 hr prior to incubation with antibodies diluted in blocking buffer. After overnight incubation in primary antibody, blots were washed several times in PBS before incubation with secondary antibody for approximately 1 h. After several PBS washes, blots were incubated with substrate (SuperSignal West Pico, Thermo) and digitally imaged on a Bio-Rad ChemiDoc imaging system.

Gumerson J.D., Alsufyani A., Yu W., Lei J., Sun X., Dong L., Wu Z, & Li T. (2021). Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing. Gene Therapy, 29(1-2), 81-93.

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Immunoblotting for TDO2 Protein Expression

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Tissues were homogenized in RIPA buffer (Nacalai Tesque) supplemented with protease inhibitor cocktail (Nacalai Tesque). Protein concentrations were measured by bicinchoninic acid assay (Thermo Fischer Scientific), and 30 µg protein was loaded per lane and separated by SDS‐PAGE electrophoresis, followed by trans blotting to PVDF membranes. The membranes were blocked with StartingBlock (PBS) Blocking Buffer (Thermo Fischer Scientific) and immunoblotted overnight with either anti‐TDO2 polyclonal Ab (#MBS9133230; MyBioSource) or anti‐β‐actin Ab (#CST4967; Cell Signaling Technology). The immobilized Ab was detected using the HRP‐conjugated secondary Ab (Cell Signaling Technology) and Chemi‐Lumi One Super (Nacalai). The images were visualized by Amersham Imager 600 (GE Healthcare).

Hagiwara A., Nakamura Y., Nishimoto R., Ueno S, & Miyagi Y. (2020). Induction of tryptophan hydroxylase in the liver of s.c. tumor model of prostate cancer. Cancer Science, 111(4), 1218-1227.

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Protein Expression Profiling of Human Glioma Cells

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Total proteins were extracted from human glioma cells using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and heated for 5 min. 4–15% Mini-PROTEAN® TGX™ Precast Gels (BIO-RAD, California, USA) were used to separate the proteins which were then transferred onto PVDF membranes. The membranes were blocked for 20 min at room temperature using StartingBlock™ (PBS) Blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 4 °C overnight with primary antibodies including CD13 antibody (rabbit, Cat #ab108310, 1/500; Abcam, Cambridge, UK), BAX (D2E11) (rabbit, Cat#5023, 1/1000; Cell Signaling Technology, Danvers, MA, USA ), BCL-2 (mouse, Cat#15071, 1/1000; Cell Signaling Technology), NOXA (mouse, Cat#ab13654, 1/1000; Abcam), Caspase-3 (D3R6Y) (rabbit, Cat#14220, 1/500; Cell Signaling Technology) and GAPDH antibody (mouse, Cat #ab9484, 1/1500; Abcam). After washing several times with TBS-T (0.05% Tween20), the membranes were incubated with HRP-conjugated secondary antibodies (1/200; Dianova, Hamburg, Germany) for 2 h at room temperature. The blots were washed several times before the enhanced chemo-luminescence detection kit (ECL Advance) was applied and luminescence was measured.

Zhang W., Blank A., Kremenetskaia I., Nitzsche A., Acker G., Vajkoczy P, & Brandenburg S. (2024). CD13 expression affects glioma patient survival and influences key functions of human glioblastoma cell lines in vitro. BMC Cancer, 24, 369.

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Western Blot Analysis of Proteins

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After SDS-PAGE using NuPAGE 10% Novex Bis-Tris gels (Life Technologies), the proteins were transferred to nitrocellulose membranes (Qiagen) and blocked in StartingBlock PBS blocking buffer (Thermo Fisher Scientific). The membranes were incubated with appropriate primary antibody overnight, followed by four 10-min washes with Tris-buffered saline with Tween 20 and then incubation with appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. Proteins were detected using either SuperSignal West Pico chemiluminescence substrate or SuperSignal West Femto maximum sensitive substrate (Pierce).

Rink C, & Williams N. (2019). Unique Interactions of the Nuclear Export Receptors TbMex67 and TbMtr2 with Components of the 5S Ribonuclear Particle in Trypanosoma brucei. mSphere, 4(4), e00471-19.

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Leptospiral Protein and LPS Separation and Analysis

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Leptospires (mid-late log phase, 1–3 × 10⁸ leptospires/ml) were harvested by centrifugation (10,000 × g, 4°C, 30 min), washed twice with Phosphate buffered saline (PBS), and processed for one-dimensional (1-D) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide gels (BioRad, Hercules, CA, U.S.A.) as per the guidelines by the manufacturer. Proteins were visualized by staining with Sypro Ruby (Invitrogen) and LPS was visualized by staining with Pro-Q Emerald 300 (Invitrogen) as per manufacturer's guidelines. For immunoblotting, samples were transferred by semi-dry transfer (Amersham TE77 PWR) to Immobilon-P transfer membrane (Millipore, 220 Bedford, MA, U.S.A.) and blocked overnight at 4°C with Starting Block (PBS) blocking buffer (Thermo Fisher). Membranes were individually incubated with indicated antisera diluted in blocking buffer (anti-LipL32 at 1:4,000, or anti-Tarassovi, anti-Hardjo, or anti-Ballum at 1:1,000) followed by incubation with horseradish-peroxidase anti-rabbit immunoglobulin G (Sigma, St. Louis, MO, U.S.A) conjugate diluted 1:4,000 in blocking buffer. Bound conjugates were detected using Clarity Western ECL substrate (BioRad) and images were acquired using a Bio-Rad ChemiDoc MP imaging system.

Hamond C., LeCount K., Putz E.J., Bayles D.O., Camp P., Goris M.G., van der Linden H., Stone N.E., Schlater L.K., Sahl J.W., Wagner D.M, & Nally J.E. (2022). Bovine Leptospirosis Due to Persistent Renal Carriage of Leptospira borgpetersenii Serovar Tarassovi. Frontiers in Veterinary Science, 9, 848664.

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Electrochemical Immunosensing with Functionalized MWCNTs

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Multiwalled carbon nanotubes
(MWCNTs), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), potassium ferricyanide, potassium
ferrocyanide, potassium chloride, disodium phosphate, trisodium phosphate,
and nitric acid were purchased from MilliporeSigma (Canada). Human
plasma fibronectin purified protein in 150 mM sodium chloride and
10 mM sodium phosphate (pH = 7.5) was obtained from MilliporeSigma
(Canada). StartingBlock (PBS) blocking buffer was obtained from Thermo
Fisher Scientific (Canada). TOP-10 Escherichia coli was supplied by courtesy of Dr. Yang Qu at the University of New
Brunswick. All chemical solids used during experimentation were measured
using Mettler Toledo balances. All electrochemical measurements were
carried out using CHI660E and CHI760E potentiostats, purchased from
CH Instrumentation (USA). Carbon screen-printed electrodes (SPEs)
were obtained from Pine Research Instrumentation (USA). Carboxyl-functionalized
MWCNT-coated carbon SPEs were acquired from Metrohm DropSens (cat.
110CNT) for use in a flow-cell format. The electrochemical flow cell
(model DRP-CFLWCL-MAGN) was purchased from Metrohm Canada and connected
to a peristaltic pump (model MasterFlex L/S) from Cole-Parmer (USA).

Flynn C.D., Sandomierski M., Kim K., Lewis J., Lloyd V, & Ignaszak A. (2023). Electrochemical Detection of Borrelia burgdorferi Using a Biomimetic Flow Cell System. ACS Measurement Science Au, 3(3), 208-216.

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SDS-PAGE and Immunoblotting of Leptospires

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Leptospires (mid-late log phase, 1–3 × 10⁸ leptospires/mL) were harvested by centrifugation (10,000 × g, 4°C, 30 min), washed twice with PBS, and processed for one-dimensional (1-D) SDS-PAGE on 12% acrylamide gels (BioRad) as per manufacturer's guidelines. Proteins were visualized by staining with Sypro Ruby (Invitrogen, CA, USA) and lipopolysaccharide was visualized by staining with Pro-Q Emerald 300 (Invitrogen, CA) as per manufacturer's guidelines. For immunoblotting, samples were transferred by semi-dry transfer (Amersham TE77 PWR) to Immobilon-P transfer membrane (Millipore, 220 Bedford, MA) and blocked overnight at 4°C with Starting Block (PBS) blocking buffer (Thermo Scientific, CO) (25 (link)).
Membranes were individually incubated with indicated antisera diluted in blocking buffer (anti-LipL32 at 1:4,000, or anti-Alexi, anti-Hardjo at 1:1,000) followed by incubation with horseradish-peroxidase anti-rabbit immunoglobulin G conjugate diluted 1:4,000 in blocking buffer (Sigma, MO). Bound conjugates were detected using Clarity Western ECL substrate (BioRad, CA) and images acquired using a Bio-Rad ChemiDoc MP imaging system.

Hamond C., Dirsmith K.L., LeCount K., Soltero F.V., Rivera-Garcia S., Camp P., Anderson T., Hicks J.A., Galloway R., Sutherland G., Schafer I.J., Goris M.G., van der Linden H., Stuber T., Bayles D.O., Schlater L.K, & Nally J.E. (2022). Leptospira borgpetersenii serovar Hardjo and Leptospira santarosai serogroup Pyrogenes isolated from bovine dairy herds in Puerto Rico. Frontiers in Veterinary Science, 9, 1025282.

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Immunohistochemical Labeling of Neuronal Nuclei

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Slices at varying depths down the UEA shank were selected for tissue staining. Tissue slices were blocked and permeabilized with a mixture of StartingBlock PBS Blocking Buffer (37538, Thermo Scientific, Waltham, MA) and 1% Triton X-100 (9002-93-1, Sigma Aldrich, St. Louis, MO) overnight at 4 °C followed by three 30-minute washes in 1X PBS containing 0.5% Triton X-100, referred to as 0.5% PBST, at room temperature. The tissue was incubated with primary antibody at a 1:250 dilution in 0.5% PBST with 0.02% sodium azide for 48 hours at 4 °C. The following primary antibody was used to stain for neurons, mouse anti-neuronal nuclei (NeuN, MAB377, MilliporeSigma, Burlington, MA). Primary antibody incubation was followed by three 30-minute washes in 0.5% PBST at room temperature. The tissue was incubated in secondary antibody at a 1:250 dilution in 0.5% PBST with 0.02% sodium azide for 24 hours at 4 °C. The following secondary antibody was used, anti-mouse Alexa Fluor 647 (715-605-150, Jackson ImmunoResearch Laboratories, Inc., Carlsbad, CA). Finally, the tissue slices were washed in room temperature 0.5% PBST two times at two-hour intervals and kept in 1X PBS overnight. All slices were stored at 4 °C in 1X PBS with 0.02% sodium azide until imaged.

Patel P.R., Welle E.J., Letner J.G., Shen H., Bullard A.J., Caldwell C.M., Vega-Medina A., Richie J.M., Thayer H.E., Patil P.G., Cai D, & Chestek C.A. (2023). Utah Array Characterization and Histological Analysis of a Multi-Year Implant in Non-Human Primate Motor and Sensory Cortices. Journal of neural engineering, 20(1), 10.1088/1741-2552/acab86.

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Immunoblotting Protein Expression Confirmation

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Immunoblotting was performed on the same samples as the SEAP assay to confirm protein expression. Briefly, cells were lysed directly in NuPAGE LDS Sample Buffer (4X; Novex), supplemented with 0.1 M DTT, boiled for 10 min at 95 °C, resolved on a 4–20% gradient gel (Bio-Rad), transferred using a semidry system (Bio-Rad) at constant current of 200 mA for 1 h, blocked using StartingBlock PBS blocking buffer (Thermo Scientific), probed using mouse monoclonal anti-FLAG (M2; 1:1,000; Sigma) overnight at 4 °C, then after washing, probed with a goat anti-mouse HRP-conjugated secondary antibody (Cusabio; 1:10,000), and visualized on an ImageQuant LAS 4000 (Cytiva) using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific).

Fiddaman S.R., Vinkler M., Spiro S.G., Levy H., Emerling C.A., Boyd A.C., Dimopoulos E.A., Vianna J.A., Cole T.L., Pan H., Fang M., Zhang G., Hart T., Frantz L.A, & Smith A.L. (2021). Adaptation and Cryptic Pseudogenization in Penguin Toll-Like Receptors. Molecular Biology and Evolution, 39(1), msab354.

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Western Blot Protein Expression Analysis

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Cells were lysed in ice-cold RIPA buffer containing proteases inhibitor cocktail (Sigma), and protein concentration was determined using the Amido Black assay. Equal amounts of protein were loaded and resolved on 10% SDS-PAGE gels, followed by transfer on 0.45 µm nitrocellulose membrane and Ponceau S reversible staining. Following incubation with StartingBlock PBS blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA), the membranes were incubated with anti-Integrin alpha5 antibody (Thermo Fisher Scientific PA5-12507), anti-Connexin 43 (Millipore AB1727), or anti-beta Actin (Sigma A2228) over-night at 4 °C. Membranes were rinsed in Tris-buffered saline solution and incubated with HRP-conjugated F(ab’)₂ fragment secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Protein bands were imaged after enhancement with chemiluminescence agent (Thermo Fisher Scientific, Waltham, MA, USA) using an ImageQuant LAS4000 system (Fujifilm, Japan). The protein expression was quantified by densitometry with ImageJ software and normalized to beta actin or total protein as quantified by Ponceau S stain.

Popescu S., Preda M.B., Marinescu C.I., Simionescu M, & Burlacu A. (2021). Dual Stem Cell Therapy Improves the Myocardial Recovery Post-Infarction through Reciprocal Modulation of Cell Functions. International Journal of Molecular Sciences, 22(11), 5631.

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