Pierce coomassie protein assay

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Pierce Coomassie protein assay is a colorimetric assay used to quantify total protein concentration in a sample. The assay utilizes Coomassie dye, which undergoes a color change when bound to proteins, allowing for the determination of protein levels through spectrophotometric analysis.

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Lab products found in correlation

Iodoacetamide, by Thermo Fisher Scientific (2 mentions) Tris 2 carboxyethyl phosphine tcep, by Thermo Fisher Scientific (1 mentions) Sequencing grade modified trypsin, by Promega (1 mentions) Ge 130 ultrasonic processor, by GE Healthcare (1 mentions) Gradient master, by BioComp Instruments (1 mentions)

Close spelling variants of this product

Pierce Coomassie Plus colorimetric protein assay Pierce Coomassie plus protein assay reagent Pierce Coomassie Plus Protein Assay Pierce Coomassie (Bradford) Protein Assay Kit Pierce Coomassie Plus (Bradford) Protein‐Assay Pierce Coomassie (Bradford) Protein Assay Pierce Coomassie Protein Assay Kit Pierce™ Coomassie plus protein assay kit Coomassie protein assay Protein assay

4 protocols using pierce coomassie protein assay

Characterization of Birch Pollen Extract

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Commercial Betula verrucosa/pendula pollen (Batch 012517101 Allergon, Angelholm, Sweden) was dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS) (ratio m/v = 0.18 g/ml) and shaken at room temperature (RT) for 1 h. BPE was collected by centrifugation for 15 min at 12,000 g, subsequently filtered through a 0.45 μm syringe filter (Merck Millipore, Merck KGaA, Darmstadt, Germany) and stored at −20°C. A single, carefully characterized BPE batch was used throughout the study. The herein determined levels of TLR4 activity/corresponding endotoxin concentrations were in a similar range to previously published studies using different pollen sources and/or different pollen batches (28 (link), 29 (link), 57 (link), 58 (link)).
The total protein concentration was determined using the Bradford assay (Pierce Coomassie Protein assay, Thermo Scientific, Waltham, MA, USA) and a bovine serum albumin (BSA) standard. BPE had a concentration of 2,200 μg/ml. The protein profile was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, as described (59 (link)).

Pointner L., Kraiem A., Thaler M., Richter F., Wenger M., Bethanis A., Klotz M., Traidl-Hoffmann C., Gilles S, & Aglas L. (2021). Birch Pollen Induces Toll-Like Receptor 4-Dependent Dendritic Cell Activation Favoring T Cell Responses. Frontiers in Allergy, 2, 680937.

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Cryogrinding and Trypsin Digestion of Frozen Mouse Heart Tissue

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Frozen cross-linked mouse heart tissue was transferred with the aid of 0.5 mL of 0.1 M NH₄HCO₃, to a stainless steel cryogrinding jar cooled to −196°C with liquid nitrogen. Using a Retsch MM 400 mixer mill, the sample was cryoground for five three minute cycles at 30 Hz and cooled with liquid N2 between cycles. The resulting frozen powder was transferred to a 15 mL tube and 1 mL of 8 M urea in 0.1 M tris pH 8.0 was added. Sample viscosity was reduced by sonication using a GE – 130 ultrasonic processor, followed by reduction and alkylation of cysteine residues by incubation with 5 mM tris(2-carboxyethyl)phosphine (TCEP) (Fisher Scientific) for 30 minutes followed by a 45 minute incubation with 10 mM iodoacetamide (Fisher Scientific). To reduce the urea concentration to less than 1 M the samples were diluted by a factor of 10 with fresh 0.1 M tris buffer pH 8.0. The protein concentration was measured using the Pierce Coomassie protein assay (Thermo). A fraction of the total protein content (0.37 mg) was set aside for generation of a stage 1 database (described below) while the rest of the protein in the sample was digested with a 1:200 ratio of sequencing grade modified trypsin (Promega) to protein and was incubated at 37°C for 18 hrs.

Chavez J.D., Lee C.F., Caudal A., Keller A., Tian R, & Bruce J.E. (2017). Chemical cross-linking mass spectrometry analysis of protein conformations and supercomplexes in heart tissue. Cell systems, 6(1), 136-141.e5.

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Cryogenic Protein Extraction and Preparation

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Frozen fractions were transferred to a stainless-steel cryogrinding jar cooled to −196°C with liquid nitrogen in 0.1M NH₄HCO₃. The samples were cryoground for five 3 min cycles at 30 Hz using a Retch MM 400 mixer mill. Between cycles, the cryogrinder was cooled with liquid nitrogen. The resulting frozen powder was transferred to a falcon tube where 8M urea (in 0.1M Tris, pH 8.0) was added. Samples were sonicated using a GE-130 ultrasonic processor, followed by reduction of cysteine residues by incubation with 5mM Tris (2-carboxyethyl) phosphine (TCEP, Fisher Scientific) for 30 min, followed by alkylation with 45 min incubation with 10mM iodoacetamide (Fisher Scientific). Urea concentration was reduced to less than 1M by diluting the samples by a factor of 10 with fresh 0.1M Tris buffer (pH 8.0). The protein concentration was measured using the Pierce Coomassie protein assay (Thermo Scientific).

Caudal A., Tang X., Chavez J.D., Keller A., Mohr J.P., Bakhtina A.A., Villet O., Chen H., Zhou B., Walker M.A., Tian R, & Bruce J.E. (2022). Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart. Nature cardiovascular research, 1(9), 855-866.

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Protein Crosslinking and Fractionation

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GraFix crosslinking was carried out as previously described (Kastner et al., 2008) . Briefly, 4 mL gradients of 5%-20% glycerol and 0%-0.075% glutaraldehyde in a uniform background of the final purification buffer containing the required detergent were prepared using a Gradient Master (Biocomp). 200 mL each of a cushion of 2.5% glycerol in buffer and the purified protein sample were then applied to the top of the gradient. Gradients were then subjected to centrifugation at 215,000 xg for 18 h. 200 mL fractions were extracted from the top of the gradient, quenched with 50 mM Tris (pH 7.5) and subjected to a Pierce Coomassie Protein Assay (Thermo Fisher) to identify protein-containing fractions.

McDowell M.A., Heimes M., Fiorentino F., Mehmood S., Farkas Á., Coy-Vergara J., Wu D., Bolla J.R., Schmid V., Heinze R., Wild K., Flemming D., Pfeffer S., Schwappach B., Robinson C.V, & Sinning I. (2020). Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex. Molecular cell, 80(1).

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