Hela cells in 6-well plates were washed three times with PBS and then lysed by radio-immune precipitation assay (RIPA) with lysis buffer(1% Triton X-100, 1% deoxycholate, 0.1% SDS) (Beyotime, China).The protein concentration was quantified by BCA Protein Assay Kit (Beyotime,China) and the total proteins were subjected to SDS-PAGE and then transferred to a polyvinylidenefluoride (PVDF) membrane (Millipore, USA). After blocking with 5% skim milk or 5% Bovine serum Albumin (BSA) in TTBS buffer (1L TTBS contains 8.77g NaCl, 2.42g Tris, 1g Tween, pH7.5) for 2 hours, the membrane was incubated with primary antibodies overnight at 4°C before it was washed with 1xTTBS for 3 times, then it was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour followed by wash with 1xTTBS for 3 times, 10 minutes each. Protein bands were detected by enhanced chemiluminescence (ECL) kit (Millipore, USA) and visualized by LAS4000 mini (GE, USA). The primary antibodies used in this study including: mouse anti-flag antibody, mouse anti-GAPDH antibody and mouse anti-β-Actin (Abbkine, Redlands, CA), rabbit anti-STAT1, rabbit anti-Phospho-STAT1 (Y701) (Cell Signaling Technology, Inc., Beverly, MA). The secondary antibodies were HRP-conjugated ECL goat anti-rabbit IgG, or HRP-conjugated ECL sheep anti-mouse IgG (Abmart, China).
Mouse anti β actin
Manufactured by Abbkine
Sourced in China, United States
Mouse anti-β-actin is a primary antibody that recognizes the β-actin protein, a ubiquitous cytoskeletal protein found in all eukaryotic cells. It is commonly used as a loading control or reference protein in Western blotting and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Mouse anti flag antibody, by Abbkine (1 mentions)
Mouse anti gapdh antibody, by Abbkine (1 mentions)
Las 4000 mini, by GE Healthcare (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Mouse anti gst, by Cell Signaling Technology (1 mentions)
Rabbit anti laminb1, by Proteintech (1 mentions)
Rabbit anti nfat1, by Cell Signaling Technology (1 mentions)
Ripa lysis solution, by Applygen (1 mentions)
Proteinase inhibitor, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Abbkine (1 mentions)
Rabbit anti lc3, by Merck Group (1 mentions)
Rabbit anti p62, by Cell Signaling Technology (1 mentions)
Rabbit anti parp 1, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
4 protocols using mouse anti β actin
1
Western Blot Analysis of HeLa Cells
2
Western Blot Analysis of Cellular Proteins
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Cells were lysed in 1% Triton X-100 lysis buffer. The total protein
concentrations in the lysates were determined using a BCA protein assay kit.
Proteins from each sample were separated via 10% SDS-PAGE and
transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5%
non-fat milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST) at
37°C for 2 hours. Immunoblot analysis was performed with mouse anti-β-actin
(#A01010, Abbkine, Wuhan, China) and rabbit anti-LCP2 (#4958, Cell Signaling
Technology, Danvers, MA, USA) at 4°C for 12 hours. Membranes were then washed
with TBST buffer three times, followed by incubation with HRP-conjugated
polyclonal secondary antibodies for 1 hour at 37°C. Immunoreactive bands were
developed using the enhanced plus chemiluminescence assay (Pierce Biotechnology,
Waltham, MA, USA) following the manufacturer’s instructions. Finally, images
were analyzed using a Chemidoc XRS + System (Bio-Rad Laboratories, Hercules, CA,
USA). All experiments were repeated twice.
concentrations in the lysates were determined using a BCA protein assay kit.
Proteins from each sample were separated via 10% SDS-PAGE and
transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5%
non-fat milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST) at
37°C for 2 hours. Immunoblot analysis was performed with mouse anti-β-actin
(#A01010, Abbkine, Wuhan, China) and rabbit anti-LCP2 (#4958, Cell Signaling
Technology, Danvers, MA, USA) at 4°C for 12 hours. Membranes were then washed
with TBST buffer three times, followed by incubation with HRP-conjugated
polyclonal secondary antibodies for 1 hour at 37°C. Immunoreactive bands were
developed using the enhanced plus chemiluminescence assay (Pierce Biotechnology,
Waltham, MA, USA) following the manufacturer’s instructions. Finally, images
were analyzed using a Chemidoc XRS + System (Bio-Rad Laboratories, Hercules, CA,
USA). All experiments were repeated twice.
3
Western Blot Analysis of Protein Targets
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The cells and tissues were lysed with an appropriate amount of RIPA lysis solution (Applygen, China) containing protease inhibitors, PMSF, and β-ME. The proteins were separated by SDS-PAGE and then transferred to PVDF membranes for primary antibodies incubation and secondary antibodies incubation. Mouse anti-GFP and mouse anti-β-actin were purchased from Abbkine. Rabbit anti-calcineurin A, mouse anti-GST, and rabbit anti-NFAT1 were purchased from Cell Signalling Technology. Rabbit anti-lamin B1 was provided by Proteintech. Goat anti-mouse IgG and goat anti-rabbit IgG secondary antibodies were purchased from SGB-BIO. After the secondary antibody incubation was completed, ECL luminescent solution (Tanon, China) was used for colour development. A Tanon instrument was used to obtain images, and ImageJ was used for image analysis.
4
Protein Extraction and Subcellular Fractionation
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Total proteins were extracted with RIPA lysis buffer (Beyotime) containing 1% proteinase inhibitors (Beyotime). The mitochondria isolation followed the instructions of the Cell Mitochondria Isolation Kit (Beyotime). Nuclear and cytoplasmic proteins were isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). The protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime). Equal amounts of protein samples were subjected to 10–12% SDS-PAGE separation and then transferred to the polyvinylidene difluoride membrane (Millipore, Boston, MA, USA). Subsequent procedures were performed as described previously [38] (link). The following antibodies were used: rabbit anti-PARP-1 (1: 500, Santa Cruz, CA, USA), rabbit anti-AIF (1:1000, Abcam), rabbit anti-LC-3 (1:1000, Sigma-Aldrich), rabbit anti-p62 (1:1000, Cell Signaling Technology, MA, USA), mouse anti-PAR (1:1000, Enzo Life Sciences), mouse anti-β-actin (1:5000, Abbkine), rabbit anti-COX Ⅳ (1:1000, Proteintech, Wuhan, China), mouse anti-Histone H3 (1:3000, Abbkine), HRP-conjugated secondary antibodies (1:5000, Abbkine). Protein bands were detected by enhanced chemiluminescence kit (Thermo, USA).
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