Bovine serum albumin (bsa)

Manufactured by PerkinElmer

Sourced in Germany

Bovine serum albumin is a protein derived from bovine (cow) blood serum. It is commonly used as a laboratory reagent and standard in various biochemical and cell-based assays.

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Lab products found in correlation

Whatman, by Cytiva (2 mentions) 3h sam, by PerkinElmer (1 mentions) Ls6500 scintillation counter, by Beckman Coulter (1 mentions) Gf c filter, by Cytiva (1 mentions) Victor3, by PerkinElmer (1 mentions) Bca method, by Thermo Fisher Scientific (1 mentions) 3h ym 09151 2, by PerkinElmer (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Typhoon imager, by GE Healthcare (1 mentions) 3h alanine, by PerkinElmer (1 mentions) 3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions) Multidrop combi dispenser, by Thermo Fisher Scientific (1 mentions) Echo liquid handler, by Beckman Coulter (1 mentions) Microbeta trilux, by PerkinElmer (1 mentions) Gtpγs, by PerkinElmer (1 mentions)

8 protocols using bovine serum albumin (bsa)

Protein Methyltransferase Assays in Vitro

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The procedures for cloning, expression and purification of proteins and protein complexes used for in vitro studies (enzymology, SEC-MALLS and X-ray crystallography) are described in supplementary materials. The tRNAs from S. cerevisiae or H. volcanii strains were purified as previously described (47 (link)).
The MTase assays were performed in a total volume of 10 μl (for HvoMtq2-Trm112) or 20 μl (for HvoTrm9–Trm112) containing 400 mM potassium phosphate buffer pH 7.5, 3 M KCl, 2.5 mM EDTA, 5 mM MgCl₂, 5 mM NH₄Cl, 0.25 mg/ml Bovine Serum Albumin, 50 μM S-adenosyl-l-methionine (SAM; containing 0.87 Ci/mmol of [³H]-SAM; Perkin Elmer) and 5 pmol of HvoMtq2–Trm112 complex or 2 pmol of HvoTrm9–Trm112 complex. The reaction was initiated by adding 100 pmol of substrate (HvoaRF1, HvoaRF3 or total tRNAs) to the mixture. After incubation for 2 h at 45°C, the reaction was stopped by precipitation with cold trichloroacetic acid (5%), followed by filtration on Whatman GF/C filters. The [³H] incorporation was measured using a Beckman Coulter LS6500 scintillation counter.

van Tran N., Muller L., Ross R.L., Lestini R., Létoquart J., Ulryck N., Limbach P.A., de Crécy-Lagard V., Cianférani S, & Graille M. (2018). Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes. Nucleic Acids Research, 46(16), 8483-8499.

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Quantifying Cytoskeletal Nitrotyrosine

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18 h after drug addition, the media in the wells were aspirated and cells were incubated with 100 μl/well of cytoskeleton isolation buffer (90 mM MES pH 6.7; 1 mM EGTA; 1 mM MgCl₂; 10% (v/v) glycerol, 0.5% (v/v) Triton X-100) for 3 minutes 37°C. Cells were fixed for 10 minutes with 3.7% paraformaldehyde in PBS-0.05% Tween-20. After 3 washes with 200μl PBS -0.05% Tween-20, cells were incubated for 1 h at RT with anti nitrotyrosine antibody diluted 1:1800 in PBS-0.05% Tween-20. Cells were washed with 200 μl of PBS-0.05% Tween-20, and then incubated with Europium labelled secondary antibody diluted 1:1,000 (or otherwise as stated), in PBS-0.05% Tween-20, 1% BSA for 30 minutes. Cells were washed 5 times with 200 μl of PBS-0.05% Tween-20 followed by incubation with 100 μl per well of DELPHIA enhancement solution (Tris-HCl buffered NaCl solution pH 7.8, containing < 0.1% NaN3, bovine serum albumin, bovine gamma globulins, Tween 40, diethylenetriaminepentaacetic acid, and an inert red dye, Perkin Elmer) for 5 min at RT. Plates were read using a time-resolved fluorescence plate reader using 340 nm excitation filter and 615 nm emission filter, with a delay of 400 μs (Victor3, Perkin Elmer).

Deshpande A., Brants J., Wasylyk C., van Hooij O., Verhaegh G.W., Maas P., Schalken J.A, & Wasylyk B. (2024). TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library. PLOS ONE, 19(2), e0296960.

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Dopamine receptor binding assay

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Ventral midbrains were dissected and pooled from two mice for each sample. Samples were homogenized with a PT 1200 E polytron (Kinematica, Inc.) for 10 s on ice in 4 ml Tris buffer (50 mM Tris-HCl, 0.9% NaCl, pH 7.4 at 4 °C) and centrifuged at 30,000 g for 20 min. The pellets were resuspended in 4 ml Tris buffer, incubated for 30 min at 25 °C to release endogenous dopamine, centrifuged, and resuspended in Tris buffer. Membranes (37–68 μg of protein) were incubated in duplicate in a total reaction volume of 1 ml with 50 mM Tris containing 0.9% NaCl and 0.002% bovine serum albumin, pH 7.4, and [³H]YM-09151–2 (84.4 Ci/mmol; PerkinElmer) at 6 concentrations from approximately 0.01–0.3 nM. Nonspecific binding was determined in the presence of 1 μM spiperone. Reactions were incubated at 25 °C for 1 h and terminated by filtration through 0.05% polyethylenimine-presoaked Wallac Filtermat A filters (PerkinElmer) using a 96-well Tomtec cell harvester and ice-cold wash buffer (10 mM Tris-HCl, pH 7.4 at 4 °C, and 0.9% NaCl). Filters were allowed to dry at least 1 h before adding scintillation fluid to each filtered spot. Radioactivity on the filters was determined using a Wallac 1450 microBeta scintillation counter. Proteins were measured using the BCA method (Pierce Biotechnology).

Robinson B.G., Bunzow J.R., Grimm J.B., Lavis L.D., Dudman J.T., Brown J., Neve K.A, & Williams J.T. (2017). Desensitized D2 autoreceptors are resistant to trafficking. Scientific Reports, 7, 4379.

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ATP Hydrolysis Kinetics Assay

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The ATPase assays were carried out in 10 μL reactions in 25 mM Tris-acetate pH 7.5, 1 mM dithiothreitol, 5 mM magnesium acetate, 20 mM NaCl, 0.25 mg/ml bovine serum albumin (NEB), 150 μM unlabeled ATP, 4 nM γ-³²P-ATP (Perkin Elmer) and 200 ng of dsDNA. The reaction buffer was assembled on ice, the recombinant proteins were added, as indicated, and the reactions were incubated at 30°C for 4 h. The ATP hydrolysis was analyzed by thin layer chromatography using a standard procedure, the plates were exposed to storage phosphor screens and processed by a Typhoon Imager (GE Healthcare/Cytiva). The data were quantitated using ImageJ and plotted with Prism software.

Marsella A., Gobbini E., Cassani C., Tisi R., Cannavo E., Reginato G., Cejka P, & Longhese M.P. (2021). Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners. Cell Reports, 34(13), 108906.

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Aminoacylation Kinetics Quantification

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We carried out aminoacylation at ambient temperature in a buffer containing 50 mM Hepes (pH 7.5), 50 mM KCl, 15 mM MgCl₂, 5 mM DTT, 10 mM ATP, 0.1 mg/ml bovine serum albumin, 5 μM tRNA, 20 μM alanine (1.34 μM ³H-alanine; PerkinElmer), and 200 nM AlaRS. At various time points, we quenched reactions by spotting 10 μl aliquots of the reaction mixture onto Whatman filter pads (Maidstone) that had been presoaked in 5% trichloroacetic acid and 2 mM alanine. Before liquid scintillation counting, we washed the filter pads three times for 15 min each in ice-cold 5% trichloroacetic acid. We determined active protein concentrations by active site titration as previously described (35 (link)). We obtained aminoacylation data from three independent experiments and averaged them.

Antika T.R., Nazilah K.R., Chrestella D.J., Wang T.L., Tseng Y.K., Wang S.C., Hsu H.L., Wang S.W., Chuang T.H., Pan H.C., Horng J.C, & Wang C.C. (2023). Sequence-specific targeting of Caenorhabditis elegans C-Ala to the D-loop of tRNAAla. The Journal of Biological Chemistry, 299(9), 105149.

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Somatostatin Receptor Autoradiography

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Receptor autoradiography was performed as described by Ferone et al. 1999 . Briefly, slide sections were preincubated for 10 min in 170 mM Tris-HCl buffer, pH 7.4, and then incubated for 60 min at room temperature (RT) in 170 mM Tris-HCl buffer containing 5 mM MgCl₂ and 1% bovine serum albumin with 0.1 nM Tyr²⁵, [¹²⁵I]-Leu⁸, and D-Trp²² (Perkin Elmer, Germany). Non-specific binding was determined using 1 μM SST14 non-labelled rat somatostatin-14 (Prospec, Israel). After incubation, slides were dipped twice in ice-cold Tris-HCl buffer and in ice-cold deionised H₂O. Finally, the sections were dried under a stream of cold air. Radiolabelled sections were exposed to Kodak Biomax XAR film (Sigma-Aldrich) for 7 days. Autoradiography images were digitised and quantified using the MCID System. Brain regions were identified according to the rat brain atlas of Paxinos and Watson (1986 ).

Faron-Górecka A., Kuśmider M., Solich J., Kolasa M., Pabian P., Gruca P., Romańska I., Żurawek D., Szlachta M., Papp M., Antkiewicz-Michaluk L, & Dziedzicka-Wasylewska M. (2018). Regulation of somatostatin receptor 2 in the context of antidepressant treatment response in chronic mild stress in rat. Psychopharmacology, 235(7), 2137-2149.

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cAMP Gs Dynamic HTRF Assay Protocol

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(using Cisbio cAMP Gs Dynamic HTRF detection kit (Cisbio GsD,
Cisbio US, Inc., Bedford, MA, USA) Orthosteric stimulator dilutions and
titrations were prepared freshly in DMSO and transferred to 384LDV
microplates. Ligands and compounds were dispensed onto dry 1536-well
plate (Corning #3725) with an Echo liquid handler (Labcyte). Frozen cell
stocks were thawed in a water bath at 37°C and immediately
diluted in stimulation buffer (HBSS (Hank’s Balanced Salt
Solution with Ca²⁺ and Mg²⁺, Gibco #24020117), 5
mM HEPES (hydroxyethyl piperazineethanesulfonic acid), 0.5 mM IBMX
(3-isobutyl-1-methylxanthine, Sigma-Aldrich) and 0.075% BSA (7.5%
DTPA-purified Bovine Serum Albumin, PerkinElmer). 5 μL of cell
suspension were added per well utilizing a Multidrop Combi dispenser
(Thermo Fisher Scientific). The microplate was centrifuged at 1000 rpm
for 1 min, covered with a lid and kept at room temperature (RT) for 30
min. cAMP standard dilutions were prepared in stimulation buffer and
added to designated wells. Detection reagents, cAMP-d2 and anti-cAMP
cryptate, were diluted in cAMP detection buffer and 4 μL were
added per well. The microplate was centrifuged at 1000 rpm for 1 min,
covered with a lid and, after a 30 min incubation at RT, read on a
PHERAstar FSX microplate reader (BMG Labtech, Ortenberg, Germany) using
the HTRF (homogeneous time resolved fluorescence) module.

Dengler D.G., Harikumar K.G., Pollari S., Sun Q., Brown B.T., Shinoki-Iwaya A., Ardecky R., Miller L.J, & Sergienko E.A. (2021). Discovery of small molecule positive allosteric modulators of the secretin receptor. Biochemical pharmacology, 185, 114451.

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GLP-1R Activation Assay Using [35S]GTPγS Binding

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Preparation of GLP-1R HEK293 cell membranes and measurement of GLP-1R activation via [³⁵S]GTPγS binding to Gα_s using an antibody capture scintillation proximity assay were performed as described previously (20 (link)). Briefly, reactions contained 5 μg of membrane in 20 mm HEPES, pH 7.4, 50 mm NaCl, 5 mm MgCl₂, 40 μg/ml saponin, 0.1% bovine serum albumin, and 500 pm³⁵S-labeled GTPγS (PerkinElmer Life Sciences). Peptides and compounds were diluted and treated at a final concentration of 1% DMSO. Binding was induced for 30 min at room temperature before solubilization with 0.2% Nonidet P-40 detergent, rabbit anti-Gα_s polyclonal antibody, and 0.5 mg of anti-rabbit polyvinyl toluene beads (PerkinElmer Life Sciences). The detection mixtures were developed for 30 min, centrifuged at 80 × g for 10 min, and counted for 1 min/well using a MicroBeta TriLux instrument (PerkinElmer Life Sciences).

Bueno A.B., Showalter A.D., Wainscott D.B., Stutsman C., Marín A., Ficorilli J., Cabrera O., Willard F.S, & Sloop K.W. (2016). Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles. The Journal of Biological Chemistry, 291(20), 10700-10715.

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