Cells were washed by dPBS twice, then lysed with 25-gauge syringe needle in a lysis buffer containing 10 mM Tris-Cl pH 8, 0.5 mM CaCl2, 100 mM KCI, 20 mM EDTA, 0.5 mM ATP, 1% Triton X100, 0.5% NP-40, mammalian protease inhibitor cocktail (Sigma-Aldrich), and 0.1 mM PMSF. The mixture was incubated at 4°C for 30 min with occasional vortexing. R-actin was immunoprecipitated using a polyclonal anti-β- actin arginylated (N-terminal) antibody that reacts with the N-terminal region of mouse R-actin (catalog no. ABT 264, Millipore). The antibody–protein complexes were pulled down using protein A beads (catalog no. 15918-014, Invitrogen) and washed three times with lysis buffer. The beads were resuspended in 2× SDS-loading buffer and boiled for 10 min. The resulting protein samples were analyzed by SDS-PAGE.
Abt264
Manufactured by Merck Group
Sourced in United States
The ABT264 is a laboratory instrument designed for the analysis and detection of various biological and chemical samples. It utilizes advanced spectroscopic techniques to provide accurate and reliable measurements. The core function of the ABT264 is to facilitate the characterization and quantification of analytes in a wide range of research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
R actin, by Merck Group (2 mentions)
Anti doublecortin antibody, by Abcam (2 mentions)
Mabs436, by Merck Group (2 mentions)
Anti β 3 tubulin, by R&D Systems (2 mentions)
Rhodamine phalloidin, by Merck Group (2 mentions)
Mammalian protease inhibitor cocktail, by Merck Group (1 mentions)
Protein a bead, by Thermo Fisher Scientific (1 mentions)
α sma, by Merck Group (1 mentions)
Alexa fluor 555 conjugated anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mounting media, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Ultra 888 emccd camera, by Oxford Instruments (1 mentions)
6 protocols using abt264
1
Immunoprecipitation of Arginylated R-Actin
2
Liver Protein Extraction and Western Blot
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The liver tissue samples were homogenized in lysis buffer (1% Triton X-100, 20 mM Tris, 40 mM NaF, 0.2% SDS, 0.5% deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 100 mM NaCl, pH 7.5) and then centrifuged for 15 minutes at 15,000× g to obtain the liver homogenates. β-actin (anti-β-actin polyclonal antibodies, ABT264, Millipore, Temecula, CA, USA) and α-SMA (rabbit anti-αSMA monoclonal antibodies, Millipore, Temecula, CA, USA) antibodies were used for Western blotting.
3
Arginylated β-actin Antibody Protocol
4
Dual Immunofluorescence Staining of Actin
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Cells cultured on coverslips were fixed by 4% paraformaldehyde (PFA) at 37 °C for 30 min, followed by washing with PBS three times, and then permeabilized for 5 minutes with cold methanol (−20 °C). Then the cells were washed with PBS three times followed by sequential incubation for 60 minutes in a blocking solution (5% goat serum in PBS), then with rabbit anti-R-actin (ABT264 EMD Millipore, 1:200) for 60 min at room temperature (~23 °C), and then with mouse anti-β-actin (Clone 4C2, EMD Millipore, 1:200) for 60 min at room temperature. Cells were washed with PBS for three times, then incubated for 1 hour at room temperature with Alexa Fluor 488-conjugated anti-mouse antibody and Alexa Fluor 555-conjugated anti-rabbit antibody (Life Technologies). After the staining, coverslips with cells were first incubated in PBS with DAPI for 5 min, then washed with PBS two times followed by mounting with ProLong Diamond anti-fade mounting media (Life Technologies). All images were acquired on a Nikon Ti microscope with Andor iXon Ultra 888 EMCCD camera and analyzed by MetaMorph software.
5
Immunofluorescence Antibody Validation
6
Immunofluorescence Antibody Validation
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Rat monoclonal ATE1 antibody (EMD Millipore, MABS436) was previously described in Wang et al. (2011) (link). Rabbit polyclonal antibody to arginylated actin (R-actin) was obtained from EMD Millipore (ABT264) as part of their product validation study. Batch 13 of the antibody, not currently licensed for commercial use, is shown in the majority of the study. Anti-Doublecortin antibody (ab18723 dilutions: 1:200 for immunofluorescence) was purchased from Abcam. Mouse monoclonal anti- β-III tubulin was from R & D Systems (MAB1195, dilutions: 1:100 for immunofluorescence). Rhodamine-phalloidin was purchased from Sigma and used at the working concentration of 100 nM.
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