Bovine serum albumin (bsa)

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Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood serum. It is widely used as a protein standard and stabilizer in various biochemical and immunological applications.

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142 protocols using bovine serum albumin (bsa)

Immunofluorescence Staining of Cellular Proteins

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After treatments, the cells were suspended in 2 ml of saline A and incubated for 30 min in 35-mm tissue culture dishes containing an uncoated coverslip. Under these conditions, cells rapidly attach to the coverslip. In some experiments the cells were incubated for 5 min with the cell-permeable DNA dye (Hoechst 33342, 10 μM) prior to the end of incubation to stain the nucleus. The cells were then fixed for 1 min with 95% ethanol/5% acetic acid, washed with PBS and blocked in PBS containing bovine serum albumin (2% w/v) (30 min at room temperature).
The cells were subsequently incubated with rabbit polyclonal anti-P47 phox (sc-17844) (1:100 in PBS supplemented with 2% bovine serum albumin; Santa Cruz Biotechnology Inc. USA), rabbit polyclonal anti-Nrf2 (1:50 in PBS containing 2% bovine serum albumin; Santa Cruz Biotechnology) or monoclonal anti-cytochrome c antibody (1:100 in PBS containing 2% bovine serum albumin; Santa Cruz Biotechnology) stored for 18 h at 4° C, washed and then incubated for 3 h in the dark with fluorescein isothiocyanate (Santa Cruz Biotechnology)-conjugated secondary antibody diluted 1:100 in PBS. Stained cells were captured with a fluorescence microscope and the resulting images were processed for fluorescence determination as described above.

Fiorani M., Guidarelli A., Capellacci V., Cerioni L., Crinelli R, & Cantoni O. (2018). The dual role of mitochondrial superoxide in arsenite toxicity: Signaling at the boundary between apoptotic commitment and cytoprotection. Toxicology and applied pharmacology, 345.

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Quantifying Osteogenic Runx2 Expression

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The hBMSCs on the membranes and in the control were lysed after osteogenic differentiation for 7 days with Cell Lytic buffer for 30 min. These lysates were sonicated for 10 s and centrifuged at 14,000 rpm for 15 min at 4°C. The protein concentrations were determined using a BCA protein assay kit (Roche, Rockford, IL, USA). Equal amounts of the lysates were loaded onto a 5% stacking and 10% resolving SDS-PAGE gel. After electrophoresis, the proteins were transferred to PVDF (polyvinylidene fluoride) membranes and reacted with Runx2 (1:1,000, Abcam) antibody. For measurement, horseradish peroxidase-conjugated secondary antibodies (1:1,000; Jackson ImmunoRes, West Grove, PA, USA) were used, followed by enhanced chemiluminescence (ECL) Plus Western Blotting Detection System (GE Health-care UK Ltd, Little Chalfont, UK). The immunoreactive bands formed were quantified by scanning densitometry software (ImageJ; National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to those of a housekeeping gene, β-actin (1:1,000 dilution with 1% bovine serum albumin in 0.01 M PBS; Santa Cruz Biotechnology Inc., Dallas, TX, USA). All Western blotting analyses were repeated three times under the same conditions.

Zhou D., Qi C., Chen Y.X., Zhu Y.J., Sun T.W., Chen F, & Zhang C.Q. (2017). Comparative study of porous hydroxyapatite/chitosan and whitlockite/chitosan scaffolds for bone regeneration in calvarial defects. International Journal of Nanomedicine, 12, 2673-2687.

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Histological Kidney Analysis and Proteinuria Quantification

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Modified PLP-fixed kidney tissues were embedded into paraffin and sectioned at 5-µm thickness. Sections were stained with hematoxylin and eosin (H&E). For analysis of proteinuria, spot urine samples (2 µl) collected from each mouse at the time of sacrifice were mixed with SDS-sample loading buffer and subjected to 10% SDS-PAGE. Gels were stained with SimplyBlue^TM SafeStain (Invitrogen, Carlsbad, CA) for 1 hour and then washed with double-distilled water (ddH₂O) for 1 hour. Bovine serum albumin (Santa Cruz Biotechnology, Dallas, TX) was used for the albumin control band.

Tsuji K., Păunescu T.G., Suleiman H., Xie D., Mamuya F.A., Miner J.H, & Lu H.A. (2017). Re-characterization of the Glomerulopathy in CD2AP Deficient Mice by High-Resolution Helium Ion Scanning Microscopy. Scientific Reports, 7, 8321.

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Immunofluorescence Staining of Mouse Brain Tissues

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For immunofluorescence staining of the mice brain tissues, the isolated tissues were fixed with 4% paraformaldehyde for 24 h, then stored in 70% ethanol at 4°C. The fixed tissues were embedded in paraffin. Paraffin sections (5 μm) were blocked with PBS containing 3% bovine serum albumin (Santa Cruz Biotechnology) for 30 min at room temperature and incubated with primary antibodies followed by incubation with either anti-rabbit or mouse IgG conjugated to Alexa Fluor 488 or 594 (Thermo Fisher Scientific). For immunofluorescence staining of cultured cells, the cells were fixed with 10% formalin in PBS for 10 min, blocked with PBS containing 3% bovine serum albumin for 30 min at room temperature, then incubated with the primary antibodies followed by incubation with either anti-rabbit or mouse IgG conjugated with Alexa Fluor 488 or 594. The samples were examined by a fluorescence microscope (model BX41; Olympus) and camera (model DP80; Olympus) at room temperature.

Sai K., Nakanishi A., Scofield K.M., Tokarz D.A., Linder K.E., Cohen T.J, & Ninomiya-Tsuji J. (2023). Aberrantly activated TAK1 links neuroinflammation and neuronal loss in Alzheimer's disease mouse models. Journal of Cell Science, 136(6), jcs260102.

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Cultivation of Human Cancer Cell Lines

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Human malignant melanoma cells (SK-MEL-28, male; SK-MEL-2, sex unknown), uterine cervical cancer cells (HeLa, female) and non-small cell lung cancer cells (A549, male; H1299, sex unknown) were obtained from the Korea Cell Line Bank (Seoul, Korea). SiHa human cervical cancer cells (female) were a gift of Dr. HD Youn (Seoul National University, Seoul, Korea). HeLa, SiHa and H1299 cells were grown in Dulbecco's modified Eagle's medium (DMEM), SK-MEL-2 and A549 cells were cultured in RPMI 1640, and SK-MEL-28 cells were cultured in Minimum Essential Medium (MEM). All the media contained 10% fetal bovine serum (Welgene, Gyeongsan, Korea) and 100 units/mL of penicillin/streptomycin (Welgene), and the cells were incubated at 37°C in a 5% CO₂ incubator.
Prostaglandin E₂ (PGE2) was purchased from Cayman Chemical (Ann Arbor, MI, USA), and okadaic acid was obtained from Tocris Bioscience (Bristol, UK). Isoproterenol, H-89, dimethyl sulfoxide and 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N6-phenyladenosine-3′, 5′-cyclic monophosphate (6-Phe-cAMP) was purchased from the Biological Life Science Institute (Bremen, Germany), and bovine serum albumin was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Noh S.E, & Juhnn Y.S. (2020). Cell-type-specific Modulation of Non-homologous End Joining of Gamma Ray-induced DNA Double-strand Breaks by cAMP Signaling in Human Cancer Cells. Journal of Korean Medical Science, 35(48), e371.

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Immunofluorescence Staining of Cells

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Cells were seeded on glass coverslips in 24-well plates (Costar) at a density of 25,000 cells per well. After the incubation time, the medium was removed and cells were then fixed during 10 min with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS) before being washed three times with PBS and permeabilized with PBS-triton 1% for 5 min at room temperature. After permeabilization, cells were washed three times for 10 min with PBS-BSA 2% (Bovine Serum Albumin) (Santa Cruz Biotechnology) then incubated with the primary antibody overnight at 4 °C in the dark (Table 2). The next day, cells were washed three times with PBS-BSA 2% before being incubated for 1 h with the secondary antibody and Hoechst (Thermo Fisher Scientific H-21491) at 2 μg/mL at room temperature in the dark. Thereafter, cells were washed three times with PBS-BSA 2% and once with PBS. The coverslips were finally mounted on slides in Mowiol mounting solution (Sigma) warmed at 57 °C. Slides were observed by confocal microscopy (SP5, Leica).

Schmit K., Chen J.W., Ayama-Canden S., Fransolet M., Finet L., Demazy C., D’Hondt L., Graux C, & Michiels C. (2019). Characterization of the role of TMEM45A in cancer cell sensitivity to cisplatin. Cell Death & Disease, 10(12), 919.

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Evaluating Fas and FasL Expression in Lung Tissue

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The expression of Fas and FasL were determined via immunohistochemistry. Lung tissues were fixed in 4% paraformaldehyde for 24 h at 4°C, dehydrated, embedded in paraffin and subsequently cut into 5 mm slices. Sections were incubated with rabbit polyclonal anti-Fas (1:100; cat. no. sc-21730) and FasL (1:100; cat. no. sc-834; both Santa Cruz Biotechnology, Inc.) antibodies at 4°C for 15 h. Samples were then incubated with horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G (1:250; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 30 min at room temperature and diluted in blocking solution (5% bovine serum albumin; cat. no. B2064; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Biotin-peroxide and diaminobenzidine were used as substrates for the color reaction. The mean optical density of Fas and FasL positive cells from each section were analyzed using image cytometry with HIPAS-2000 image analysis software (Wuhan Qianli Technical Imaging Co. Ltd., Wuhan, China). The number of positive microvessels in each section was counted in 10 microscopic fields (at magnification, ×400) under a light microscope (BX51; Olympus Corporation, Tokyo, Japan). The specificity of immunohistochemical staining was tested using PBS at the same dilution. Tissue sections in the sham group were used as negative controls.

Kong Q., Wu X., Duan W., Zhan L, & Song X. (2019). Penehyclidine hydrochloride exerts protective effects in rats with acute lung injury via the Fas/FasL signaling pathway. Experimental and Therapeutic Medicine, 17(5), 3598-3606.

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Immunostaining for TLR2 and GFAP Co-Expression

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A standard technique was followed for immunohistochemistry to detect TLR2 and GFAP co-expression [17 (link)]. Formalin-fixed, paraffin-embedded tissues were sectioned at 6 μm and mounted onto positively charged glass slides. Sections were baked for overnight at 60°C, deparaffinized in xylene, and then rehydrated in graded concentrations of ethanol. Antigen retrieval was carried out for 20 min using a steamer and a citrate-based antigen unmasking solution (Vector Labs, Burlingame, CA). Tissues were blocked in blocking buffer (Dako) for one hour at room temperature before antibodies were applied. Tissues were incubated with TLR2 (ab24192, Abcam) and GFAP primary antibody (GA-5, Sigma) overnight at 4°C, washed three times with PBS with 0.2% bovine serum albumin (Santa Cruz) (PBS/BSA), and then incubated in the dark for 60 min at room temperature with secondary antibodies directly conjugated with Alexa 488 (green) or Alexa 568 (red) (Molecular Probes/Invitrogen, Carlsbad, CA). Sections were washed three times in PBS/BSA, cover-slipped with Prolong Gold with DAPI (Molecular Probes/Invitrogen), and imaged on a Nikon Eclipse TE2000-U microscope.

Inglis F.M., Lee K.M., Chiu K.B., Purcell O.M., Didier P.J., Russell-Lodrigues K., Weaver S.C., Roy C.J, & MacLean A.G. (2015). Neuropathogenesis of Chikungunya Infection: Astrogliosis and Innate Immune Activation. Journal of neurovirology, 22(2), 140-148.

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Western Blot Analysis of Protein Signaling

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Cells were lysed in RIPA lysis buffer (Thermo Scientific, Rockford, IL). Protein concentration was measured using a Bradford protein assay (Thermo Scientific, Rockford, IL). Equal amount of protein (30 μg) were separated by 8% SDS-PAGE and transferred to a PVDF membrane by electroblotting. Membranes were blocked by 5% non-fat dry milk (LabScientific, Highlands, NJ) or bovine serum albumin (Santa Cruz Biotechnology, Dallas, TX) in 0.1% Tris-buffered saline-Tween-20 (TBST) for 1 h at room temperature and incubated in TBST containing primary antibodies overnight at 4 °C. Membranes were incubated with anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Danver, MA) for 1 h at room temperature. Protein expression was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) and membranes were exposed and analyzed via Li-Cor Odyssey FC imaging system (Li-Cor, Lincoln, NE). Antibodies against p-AMPKα (Thr172), AMPKα, p-ACC (Ser79), ACC, p-LKB1 (Ser428), LKB1, p- PKCζ (Thr410), PKCζ, and p-CaMKKβ (Ser511) were used to detect target protein level at 1:1000. β-Actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Dallas, TX) was used as the loading control.

MacDonald A.F., Bettaieb A., Donohoe D.R., Alani D.S., Han A., Zhao Y, & Whelan J. (2018). Concurrent regulation of LKB1 and CaMKK2 in the activation of AMPK in castrate-resistant prostate cancer by a well-defined polyherbal mixture with anticancer properties. BMC Complementary and Alternative Medicine, 18, 188.

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Selecting gC1qR-binding Plasmodium falciparum clones

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To select for binding to gC1qR, a P. falciparum 3D7 culture synchronized in trophozoite/schizont stages was incubated for 1 h in bacteriological Petri plates coated with 2 mL of recombinant gC1qR diluted in PBS (50 μg/mL) [14 (link)]. Unbound parasites were collected using a pipette and separated from bound parasites. Both unbound and bound parasites were cultured, with the latter being subjected to a second round of selection for binding to gC1qR. After a limiting dilution cloning, a selected and unselected clone were expanded and tested for binding to gC1qR, CD36, ICAM-1, CSA (Chondroitin sulfate A sodium salt from bovine trachea Sigma-Aldrich) and BSA (Bovine Serum Albumin, Santa Cruz Biotechnology), following standard procedures [8 (link)]. The var genes transcription profile was determined for both clones by individual qPCR performed in duplicate using primers covering the P. falciparum 3D7 var gene repertoire [71 (link),76 ].

Magallón-Tejada A., Machevo S., Cisteró P., Lavstsen T., Aide P., Rubio M., Jiménez A., Turner L., Valmaseda A., Gupta H., De Las Salas B., Mandomando I., Wang C.W., Petersen J.E., Muñoz J., Gascón J., Macete E., Alonso P.L., Chitnis C.E., Bassat Q, & Mayor A. (2016). Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in Severe Malaria. PLoS Pathogens, 12(11), e1006011.

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