Protein extraction was performed using cell lysis buffer (50 mM Tris-base, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 1 mM PMSF) on ice. Equal amount of proteins were subjected to 6% SDS-PAGE and transblotted to PVDF membrane. After incubation with the blocking buffer (5% nonfat milk), the membrane was subjected for overnight incubation at 4℃ with primary antibody against MACF1 (Abcam, USA), or GAPDH (Sigma-Aldrich, USA). The horseradish peroxidase (HRP) conjugated secondary antibody was further used. Protein bands were visualized by chemiluminescence using an ECL kit (Pierce, USA) and exposed to X-ray film.
Macf1
Manufactured by Abcam
Sourced in United States
MACF1 is a protein that is involved in the organization and movement of cytoskeletal structures within cells. It plays a role in the formation and maintenance of microtubules, which are essential for various cellular processes such as cell division, intracellular transport, and cell shape.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Merck Group (1 mentions)
Lsm 5 exciter confocal microscope, by Zeiss (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Vibratome, by Campden Instruments (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fv1000, by Olympus (1 mentions)
P smad1 5 9, by Cell Signaling Technology (1 mentions)
Westernbright ecl spray, by Advansta (1 mentions)
Gapdh, by Wuhan Servicebio Technology (1 mentions)
Runx2, by Wuhan Servicebio Technology (1 mentions)
Bmp 2, by Wuhan Servicebio Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
4 protocols using macf1
1
MACF1 Protein Expression Analysis
2
Immunohistochemistry of Brain Samples
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Immunohistochemistry was performed as described previously2 (link). Briefly, animals were anaesthetised with pentobarbital and then perfused with with ice-cold PBS followed by 4% PFA in PBS. The brains were extracted and fixed in 4% PFA in PBS overnight at 4 °C. The following day, brains were washed with PBS and 70 µm thick coronal sections were cut using a vibratome (Campden Instruments, UK). Sections were blocked with 10% FBS in PBS-T (PBS + 0.1% Triton X-100) for 1 h at RT followed by incubation with primary antibodies overnight. Antibodies used were: PGAP2 (Abcam, 1:500), GPX3 (Abcam, 1:1000), MACF1 (Abcam, 1:1000), NeuN (Chemicon, 1:1000), GFAP (Abcam, 1:2000), HOMER (Synaptic Systems, 1:500). The secondary, Alexa-Fluor conjugated antibodies were chosen accordingly to wave length emission (Molecular Probes). Sections were mounted with Fluorsave medium (Calbiochem) and photographs were taken using a Zeiss LSM 5 Exciter confocal microscope.
3
Immunofluorescence Analysis of METTL14 and MACF1
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Cells were washed with phosphate-buffered saline, fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.25% Triton X-100 in phosphate-buffered saline for 5 min, followed by 1 h incubation with primary antibodies, METTL14 (Novus), MACF1 (Abcam) and then incubation with IgG (Santa Cruz). The coverslips were counterstained with 4,6-diamidino-2-phenylindole (Invitrogen) and imaged with a confocal laser-scanning microscope (Olympus FV1000, Olympus).
4
Osteoblast Protein Extraction and Analysis
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Tibia, liver, spleen and heart of the male mice were immediately separated and pulverized in a cooled mortar with liquid nitrogen. The bone marrow in tibia was flushed out before pulverization. Primary osteoblasts were washed with ice‐cold PBS before protein extraction. Whole‐cell lysates for Western blot were extracted using RIPA Lysis buffer (Beyotime) with protease and phosphatase inhibitor cocktails (Calbiochem). The concentration of protein was analysed by BCA protein assay. Protein lysates were separated using 10% SDS‐PAGE at 120V for 1 hour and then electro‐transferred to nitrocellulose membranes at 100V for 2 hours. The membranes were blocked with 5% non‐fat milk in TBST and then incubated with antibodies against Macf1 (1:500, Abcam), Alp (1:1000, Santa Cruz Biotech), Col1 (1:1000, Cusabio Technology), Bmp2 (1:1000, Servicebio Technology), p‐Smad1/5/9 (1:1000, Cell Signalling Technology), Smad1 (1:1000, Cusabio Technology), Runx2 (1:500, Servicebio Technology) and Gapdh (1:2000, Servicebio Technology) overnight at 4°C, followed by incubation with corresponding secondary antibodies for 2 hours at room temperature. Protein bands were facilitated by Western Bright ECL Spray (Advansta San Jose) and visualized by a T5200 Multi chemiluminescence detection system (Tanon). Densitometric quantification of the bands was performed with Image J analysis software (Image J).
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