Ab99046

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab99046 is a laboratory product offered by Abcam. It is a high-quality reagent designed for use in scientific research applications. The core function of this product is to serve as a tool for researchers in their experimental procedures. No further details or interpretation about its intended use are provided.

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Lab products found in correlation

6 protocols using ab99046

Antibody Validation for BKCa Channels

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An antibody against BK_Ca-α (ab99046) used for immunohistochemical staining and western blot was purchased from abcam (Cambridge, UK). An antibody against BK_Ca-β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel). Antibodies against α-SMA (ab5694), SM22α (ab10135), and eIF5 (ab228874) used for western blot were purchased from abcam (Cambridge, UK).

Wang M., Li S., Liu H., Liu M., Zhang J., Wu Y., Xiao C, & Huang H. (2022). Large-conductance Ca2 +-activated K+ channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells. Frontiers in Cardiovascular Medicine, 9, 1062695.

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Quantification of IP3R and BKCa in Aortic Lysates

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Denuded aortae were pulverized in liquid nitrogen, re-suspended in ice-cold cell lysis buffer (50 mM Tris [pH 7.4], 50 mM NaF, 1 mM Na₄PPi, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, and 1% cocktail of protease inhibitors) and protein concentrations determined using Coomassie Plus Protein Assay Reagent (Perbio, USA). Homogenates were run at several protein concentrations (2.5, 5 and 7.5 µg/lane) on NuPAGE Novex 4–12% Bis-Tris mini gels (Life Technologies) and transferred to a nitrocellulose membrane, and IP3R was detected using rabbit polyclonal anti-IP3R1 (No. 3763 used at 1:1,000; Cell Signaling Technology). Large conductance calcium-activated potassium channels (BK_Ca) in lysates were detected using an anti BK_Ca mouse monoclonal antibody (1:500 dilution, ab99046; ABcam). Secondary HRP-conjugated antibodies were either goat anti-rabbit HRP conjugated for IP3 detection (ab6721; Abcam) or rabbit anti-mouse HRP conjugated for BK_Ca detection (ab6728; Abcam). IP3R and BK_Ca protein abundance was quantified using Quantity One software (BioRad). Expression was normalized to GAPDH (1:40,000. No. G8795; Sigma-Aldrich) or α-actin (1:1,000, No. 14958; Cell Signaling Technology).

Ewart M.A., Ugusman A., Vishwanath A., Almabrouk T.A., Alganga H., Katwan O.J., Hubanova P., Currie S, & Kennedy S. (2017). Changes in IP3 Receptor Expression and Function in Aortic Smooth Muscle of Atherosclerotic Mice. Journal of Vascular Research, 54(2), 68-78.

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Immunolocalization of BK Channel Subunits

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Freshly dispersed myocytes were fixed in 4% paraformaldehyde and treated with blocking solution containing 20% goat serum, 2% bovine serum albumin, and 0.1% Triton-100 for 30 min. Slips were incubated with mouse monoclonal anti-BK α (channel-forming) subunit antibody ab99046 (Abcam, Cambridge, MA) and rabbit polyclonal anti-BK β1 subunit antibody (Thermo Scientific, Waltham, MA) at 4°C overnight. Two negative controls were used. To determine any possible background fluorescence caused by tissue natural fluorescence and by non-specific binding of secondary antibody to the specimen, we omitted addition of primary antibodies (Figs. S1–2). To confirm specificity of binding for anti-BK β1 antibody, we pre-incubated antibody with immunogenic peptide corresponding to the BK β1 subunit sequence (Waltham, MA) (Fig. S2).
The day following overnight incubation with anti-BK subunit antibodies, slips were washed and incubated with pre-absorbed Alexa-488-conjugated anti-rabbit and Cy5-conjugated anti-mouse secondary antibodies at room temperature in the dark for 2 hours. Slips were mounted using ProLong AntiFade kit (Invitrogen, Carlsbad, CA) and sealed using clear nail polish. Immunofluorescence images were obtained sequentially using 488 and 635 laser lines of Olympus FV-1000 laser scanning confocal system (Center Valley, PA).

Bukiya A., Dopico A., Leffler C, & Fedinec A. (2014). Dietary cholesterol protects against alcohol-induced cerebral artery constriction. Alcoholism, clinical and experimental research, 38(5), 1216-1226.

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Co-expression and Co-immunoprecipitation of mSlo1 and Cav1.2

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mSlo1 and C-HA tagged Ca_v1.2 in pcDNA3.1 were co-expressed in HEK293 cells, 24 hrs after transfection, the cells were lysed (lysis buffer contained 20 mM Tris-HCl/pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride and protease inhibitors. After vertical rotated at 4 °C for 1 h, the lysed cells then were high-speed centrifuged (12, 000 rpm) at 4 °C for 30 min. The supernatants were then added loading buffer and boiled at 60 °C for 10 min. Proteins in the lysate were separated on polyacrylamide gels and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk in 0.1% Tween 20 in Tris-buffered saline, the blots were probed with mouse monoclonal anti-Slo1 antibody (abcam, ab99046) and mouse monoclonal anti-human HA antibody (Millipore, 05-904), respectively. Horseradish peroxidase-coupled goat anti-mouse IgG was used as the secondary antibody for the blots. The membrane was washed with 0.1% Tween 20 in Tris-buffered saline, and proteins were visualized with an enhanced chemiluminescence detection system.

Hou P., Xiao F., Liu H., Yuchi M., Zhang G., Wu Y., Wang W., Zeng W., Ding M., Cui J., Wu Z., Wang L.Y, & Ding J. (2016). Extrapolating microdomain Ca2+ dynamics using BK channels as a Ca2+ sensor. Scientific Reports, 6, 17343.

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Immunocytochemistry Protocol for Cellular Protein Analysis

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Immunocytochemistry analysis was performed according to a previous report^{21 (link)}. The cells were seeded onto slides. At 70% confluence, the cells were fixed in 4% paraformaldehyde in PBS buffer for 15 min. Following washes with PBS, the cells were permeabilized in freshly prepared 0.1% Triton X-100 for 10 min and blocked in 1% bovine serum albumin (BSA) in PBS buffer for 1 h at room temperature. The cells were incubated with primary antibodies directed against either runx2 (1:200, ab76956, Abcam), the α-subunit of BK (1:100, ab99046, Abcam, USA), integrin beta1 (1:100, 1798-1, Epitomics, USA), Flag (1:200, Rabbit Monoclonal Anti-Flag antibody, F2555, Sigma) or His (1:200, SAB1306084, Sigma) at 4 °C overnight. The secondary Alexa Fluor 647-conjugated anti-mouse antibody (Jackson Immuno Research, USA) was used at a 1:200 dilution for 2 h at room temperature protected from light. Following washes with PBS, the cells were stained with DAPI for 5 min. The slides were mounted with SlowFade™ Gold Antifade Mountant (S36936, Invitrogen), and images were captured using a Leica TCS SP5 confocal microscope (Leica, Germany) at room temperature with ×10 and ×40 objective lenses.

Wang Y., Guo Q., Hei H., Tao J., Zhou Y., Dong J., Xin H., Cai H., Gao J., Yu K., Reilly S., Yin P, & Zhang X. (2019). BK ablation attenuates osteoblast bone formation via integrin pathway. Cell Death & Disease, 10(10), 738.

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Immunofluorescent Localization of Muscle Proteins

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Protein localization was performed in tissue sections of gracilis muscle from control rats. Briefly, gracilis muscles were fixed with 2% paraformaldehyde and embedded in paraffin for tissue sectioning. Five-micron-thick sections were blocked with 5% normal donkey serum and incubated overnight with mouse anti-BK (1:200, Abcam [ab99046]), rabbit anti-TRPV4 (1:100, Abcam [ab39260]), or goat anti-caveolin-1 (Cav-1; 1:200, Abcam [ab192452]). All sections were treated with mouse or goat anti-alpha actin (1:300). Tissue sections were then incubated with appropriate secondary antibodies for 20 min at RT. 633 hydrazide (1 μm) was used to stain the internal elastic lamina. Samples were mounted with anti-fade mounting media and images were acquired using a confocal microscope (TCS SP5; Leica Microsystems).

Naik J.S, & Walker B.R. (2018). Endothelial-dependent dilation following chronic hypoxia involves TRPV4-mediated activation of endothelial BK channels. Pflugers Archiv, 470(4), 633-648.

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