Anti rabbit

Manufactured by PerkinElmer

Sourced in United States

Anti-rabbit is a laboratory reagent used in various immunoassay techniques. Its core function is to specifically bind to rabbit-derived antibodies, enabling their detection and quantification in biological samples.

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Lab products found in correlation

4 protocols using anti rabbit

Protein Extraction and Western Blot Analysis

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Cells were plated and treated as described in section 2.3. After 24 h, culture medium was removed, and cells were washed with PBS. To obtain total protein extracts, cell lysis was performed chemically, with Lysis Buffer (5 mM Hepes pH 7.5, 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1.5 mM EGTA, 4 nM PMSF, 1% Aprotinine, 20 nM sodium pyrophosphate, 10 nM sodium orthovanadate), and mechanically, with a cell scarper.
Total protein samples were then clarified by centrifugation (13,000g, 15 min, 4 °C) and quantified using Bradford method.
10 μg of proteins were loaded in a polyacrylamide gel and after SDS-PAGE electrophoresis, western blotting was performed as described by antibodies manufacturers (anti-ERα, 1:1000, Santa Cruz, USA; anti-BRCA1, 1:50, Cell Signaling, USA; anti-CAV1, 1:1000, Cell Signaling, USA; anti-actin, 1:1000, Santa Cruz, USA; anti-LC3, 1:1000, Cell Signaling, USA; anti-Beclin1, 1:1000, Cell Signaling, USA; anti-rabbit, 1:2000, PerkinElmer, USA; anti-mouse, 1:2000, Chemicon, USA; anti-goat, 1:2000, Chemicon, USA).

Malacrida A., Erriquez J., Hashemi M., Rodriguez-Menendez V., Cassetti A., Cavaletti G, & Miloso M. (2022). Evaluation of antitumoral effect of Hibiscus sabdariffa extract on human breast cancer cells. Biochemistry and Biophysics Reports, 32, 101353.

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Western Blot Analysis of Tubulin

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Protein extracts were obtained as described in protein extracts paragraph with a complete lysis buffer.
10 µg of proteins were mixed with Laemmli buffer and then denatured at 95 °C for 5 min. Proteins were separated in a 13% acrylamide SDS-PAGE and, after electrophoresis, transferred to nitrocellulose filters for western blot analysis.
Membranes blocking, washing and antibody incubation were performed according to manufacturer’s instructions. Antibodies against β-tubulin (1:2000, Sigma-Aldrich, USA) and GTP-tubulin (1:1000, Adipogen, USA) were used. After incubation with primary antibodies, membranes were washed and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) (anti-mouse, Chemicon, USA; anti-rabbit, PerkinElmer, USA; anti-human, Sigma-Aldrich, USA). Immunoreactive proteins were visualized using an ECL chemiluminescence system (Amersham, USA).

Malacrida A., Semperboni S., Di Domizio A., Palmioli A., Broggi L., Airoldi C., Meregalli C., Cavaletti G, & Nicolini G. (2021). Tubulin binding potentially clears up Bortezomib and Carfilzomib differential neurotoxic effect. Scientific Reports, 11, 10523.

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Comparative Protein Expression Analysis of Stem Cells

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Total cellular extracts from hASCs, hS-MSCs, hPDLSCs, and hDPSCs were prepared as previously described [14 (link)]. To obtain nuclear protein extracts the Ronca et al. protocol was performed [15 (link)]. Protein concentration was determined by the Bradford assay using a Coomassie Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). After electrophoresis onto 13% SDS-PAGE, the proteins were transferred to nitrocellulose filters and immunoblotting analysis was performed using anti-βIII-tubulin (1 : 3000) and anti-NeuN (1 : 200) antibodies. Anti-actin (1 : 1000, Santa Cruz, Temecula, CA, USA) antibody was used as a loading control. After incubation with primary antibodies, the membrane was washed and then incubated with the appropriate horseradish peroxidise conjugated secondary antibodies (1 : 2000) (anti-mouse, Chemicon, Temecula, CA; anti-rabbit, PerkinElmer, Boston, MA; anti-goat, Santa Cruz Temecula, CA, USA) and the ECL chemiluminescence system (Amersham, Arlington Heights, IL, USA) was used.

Foudah D., Monfrini M., Donzelli E., Niada S., Brini A.T., Orciani M., Tredici G, & Miloso M. (2014). Expression of Neural Markers by Undifferentiated Mesenchymal-Like Stem Cells from Different Sources. Journal of Immunology Research, 2014, 987678.

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Synaptosome Purification and Protein Analysis

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Synaptosome purification has been described previously^{26 (link)}. For the validation of NBEA expression after retrieval, whole hippocampal tissue was used. For the rest of the study, the dorsal hippocampus was dissected and homogenized in ice-cold lysis buffer using a Teflon-glass tube. For immunoblotting analysis, antibodies targeting the following proteins were used. Primary antibodies against the following antigens were used: synapsin I (1:1,000, Abcam, Cambridge, UK), postsynaptic density 95 (PSD95; 1:1,000, Abcam, Cambridge, UK), histone H1 (1:1,000, Abcam, Cambridge, UK), NBEA (K-20) (1:1,000, Santa Cruz Biotech., Dallas, TX, USA), A kinase anchor protein 150 (AKAP150; 1:1,000, Santa Cruz Biotech, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000, Cell signaling, Danvers, MA, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse (1:5,000) or anti-rabbit (1:10,000) antibodies (PerkinElmer Life Sciences, Norwalk, CT, USA). Signal detection was performed using the LAS 2000 system (LAS-2000, Fuji, Tokyo, Japan) with enhanced chemiluminescence (Western-CDP star, PerkinElmer Life Sciences, Norwalk, CT, USA). Densitometric analysis of immunoreactivity for each protein was conducted using NIH ImageJ software.

Lee B., Bang E., Yang W.S., Paydar A., Ha G.E., Kim S., Kim J.H., Cho T., Lee S.E., Lee S., Kang M.G., Cheong E., Kim K.S., Lee C., Yu M.H, & Shin H.S. (2018). The Possible Role of Neurobeachin in Extinction of Contextual Fear Memory. Scientific Reports, 8, 13752.

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