The following antibodies were used in this study: CBFβ rabbit polyclonal (ab33516, Abcam, Cambridge, MA, USA) and A303-547A, A303-548A, A303-549A, Bethyl Labs, Montgomery, TX, USA). Dilutions for ab33516 were IF (1:500) and WB (1:500). Dilutions and concentration used for CBFβ Bethyl antibodies were IF (1:1000), IP (5 µg) and WB (1:1,000). GFP rabbit polyclonal (ab290, Abcam, Cambridge, MA, USA), was used at dilutions for WB (1:1000) and IP (5 µl). RUNX2 (8G5) mouse monoclonal (MBL International, Woburn, MA, USA), dilution used for IF (1:600). Filamin A (EP2405Y) rabbit polyclonal (ab76289, Abcam, Cambridge, MA, USA) dilution was used for IF (1:1000). Beta-tubulin mouse monoclonal (T-4026, Sigma Aldrich, St. Louis, MO, USA), dilution used for IF (1:1000) and WB (1:1000). PRC1 goat polyclonal (K-18) (sc-9342, Santa Cruz Biotechnology, Santa Cruz, CA, USA), dilution used for IF (1:300). KIF4A goat polyclonal (ab3815, Abcam, Cambridge, MA, USA), dilution used for IF (1:300). MRLC3 goat polyclonal (sc9449, Santa Cruz Biotechnology, Santa Cruz, CA, USA), dilution used for WB (1:200). Secondary antibodies conjugated with HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), dilution used for WB (1:5000). Secondary antibodies conjugated with Alexa fluor 488 or 594 (Life Technologies-Invitrogen, Carlsbad, CA, USA), were used at dilution IF (1:500).
Ab76289
Manufactured by Abcam
Sourced in United States
Ab76289 is an assay kit designed for the quantitative measurement of a specific target protein. The kit includes all the necessary reagents and components to perform the assay. The core function of this product is to provide a standardized and reproducible method for the quantification of the target protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab3815, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Ab33516, by Abcam (1 mentions)
Ab195380, by Abcam (1 mentions)
Ab51520, by Abcam (1 mentions)
Ab109012, by Abcam (1 mentions)
Non fat milk powder, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Ab8227, by Abcam (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
H3663, by Merck Group (1 mentions)
F1084, by Merck Group (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
β actin, by ABclonal (1 mentions)
Chemidoctm xrs system, by Bio-Rad (1 mentions)
Ab179471, by Abcam (1 mentions)
Ab96723, by Abcam (1 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
6 protocols using ab76289
1
Antibody Profiling for Cellular Analysis
2
Western Blot Analysis of Key Proteins
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The specific steps of Western blot were performed as described previously [33 (link)]. First, the radioimmunoprecipitation assay buffer (RIPA) was used to obtain proteins, followed by protein concentration assay using bicinchoninic acid (BCA) assay kit (Beyotime, China). Next, proteins were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidene (PVDF, Bio-Rad, USA) membrane. Then, 5% nonfat milk powder (Beyotime, China) was used to enclose the membrane at room temperature for 1 h. After blocking, the primary antibodies were introduced to incubate overnight at 4°C. After incubation, TBST (Beyotime, China) was used to wash the membrane, which was then incubated at room temperature with secondary antibody for 4 h. Finally, ECL hypersensitive chemiluminescence kit was used for protein color development, and pictures were taken with the visualizer. The primary antibodies were sourced from Abcam (UK): rabbit anti-human WTAP (ab195380), FLNA (ab76289), LC3I/II (LC3B, ab51520), β-actin (ab8227), p62 (ab109012). The secondary antibody was goat anti-rabbit IgG (ab6721), which was purchased from Abcam (UK).
3
Western Blotting of Human Aorta Proteins
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Western blotting was performed as previously described (14 (link)). Human aorta specimens were homogenized with RIPA lysis buffer containing protease inhibitor complex and phosphatase inhibitors, and the protein concentration was assayed using a BCA protein assay kit (23227, Thermo Fisher Scientific). Twenty micrograms of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore). The membranes were blocked with 5% nonfat milk and then incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody. Finally, the membranes were incubated in ECL reagents prior to visualization using a ChemiDocTM XRS+ system (Bio-Rad). The antibodies used in this study included FLNA (ab76289, Abcam), FLNB (GTX101206, GeneTex), FLNC (ab180941, Abcam), HA (H3663, Sigma), Flag (F1084, Sigma), and β-Actin (AC026, ABclonal).
4
Hippocampal Protein Expression Analysis
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Hippocampal tissues were homogenized in lysis buffer containing protease inhibitors and phosphatase inhibitor cocktail (p0013B, Beyotime Biotechnology, Shanghai, China). Equal amounts of protein (50 μg per line) in sample buffer were denatured at 95 °C for 5 min and separated on 10% acrylamide gel. The proteins from the gel were transferred to a nitrocellulose membrane. The membranes were incubated in 3% non-fat milk in Tris buffer for 30 min and then incubated overnight with primary antibodies against: Col1a2 (1:500; Abcam/ab96723, Cambridge, MA, USA), Flna (1:100; Abcam/ab76289, Cambridge, MA, USA), Itgb1 (1:500; Abcam/ab179471, Cambridge, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:4000; Abcam/ab181602, Cambridge, MA, USA). Subsequently, the membranes were incubated with secondary anti-rabbit (1:5000; Santa/Sc-2004, Santa Cruz, CA, USA), followed by chemiluminescent detection by enhanced chemiluminescence (Thermo/34080). The density of the bands on the membrane was scanned and analyzed with LabWorks Image Analysis Software (UVP, Inc., San Gabriel, CA, USA).
5
Multiplex Immunohistochemistry for KIRC
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Multiple immunohistochemistry (mIHC) was performed to detect three different antibodies on tissue sections. The primary antibodies used were rabbit monoclonal (1:500 dilution, ab186754, EPR15827(B); Abcam) to DSTN, rabbit monoclonal (1:200 dilution, ab76289, EP2405Y; Abcam) to FLNA, and rabbit monoclonal antibody (1:200 dilution, ab133616, EPR6855; Abcam) to CD4. The cancerous or adjacent normal tissues used for mIHC experiments were obtained from archived paraffin-embedded surgical specimens of patients with KIRC who provided prior informed consent. Employing the TSA method, the primary antibodies were stained. The procedure involved deparaffinization, antigen repair, labeling, inactivation of endogenous peroxidases, and antigen blocking on the first day. The first primary antibody, CD4, was applied, incubating overnight at 4°. On the second day, after treatment with goat antirabbit IgG (H+L; 1:50 dilution) labeled with horseradish peroxidase, Cyanine 5 Tyramide was used for detection. The steps were then repeated with DSTN or FLNA primary antibodies, incubating overnight at 4°. On the third day, following treatment with goat anti-rabbit IgG (H+L; 1:50 dilution) labeled with horseradish peroxidase, the sections were incubated with fluorescein tyramide working solution. Finally, nuclei were stained with DAPI.
6
Immunoblotting Analysis of Cellular Proteins
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SaOS2, 293T, and HeLa cell lines were cultured in six-well plates. At 90% confluence, cells were harvested and lyzed using 250 μl of 1 × SDS loading buffer. After heating for 10 min at 100 °C, 20 μl cell lysate was loaded into wells within an SDS-PAGE gel for electrophoresis. After that, immunoblotting was performed using antibodies against the factors, including FLNA (Ab76289, Abcam), TFAP2A (Ab108311, Abcam), HA (H9658, Sigma-Aldrich), GAPDH (RAB0101, Frdbrio), BRF1 (SC-81405, Santa Cruz Biotech), GTF3C2 (SC-81406, Santa Cruz Biotech), c-MYC (SC-40, Santa Cruz Biotech), MDM2 (SC-965, Santa Cruz Biotech), p53 (SC-126, Santa Cruz Biotech), RhoA (SC-418, Santa Cruz Biotech), mCherry (TAG0080, Frdbio), TBP (SC-421, Santa Cruz Biotech), and PTEN (SC7974, Santa Cruz Biotech).
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