Total lysates for the immunoblotting analyses were obtained as follows: the primary hippocampal neurons were washed in phosphate buffered saline (PBS) and collected by gentle scraping in ice-cold RIPA buffer containing (in mM): 50 Tris pH 7.4, 100 NaCl, 1 EGTA, 1 PMSF, 1 Na3VO4, 1 NaF, 0.5% NP-40, and 0.2% SDS, and supplemented with protease inhibitor cocktail II (Roche Diagnostic, Monza, Italy). The nitrocellulose membranes were incubated with the following antibodies: rabbit KV2.1 antibody (1:1000, Alomone Labs, Jerusalem, Israel), mouse α-tubulin antibody (1:5000, Sigma-Aldrich, Milan, Italy), and anti-β-actin peroxidase (1:10,000, Sigma-Aldrich, Milan, Italy). The immunoreactive bands were detected with the chemiluminescence system (GE Healthcare, Milan, Italy). The films were developed with a standard photographic procedure, and the quantitative analysis of the bands detected was carried out by densitometric scanning.
α tubulin mouse antibody
Manufactured by Merck Group
Sourced in United States
The α-tubulin mouse antibody is a primary antibody that recognizes the α-tubulin protein, a key component of the cytoskeleton in eukaryotic cells. This antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and study the distribution and expression of α-tubulin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
1.4 na oil immersion objective, by Nikon (2 mentions)
Alexa fluor 633, by Thermo Fisher Scientific (2 mentions)
A1r inverted confocal microscope, by Nikon (2 mentions)
Cocktail of protease inhibitor, by Merck Group (2 mentions)
Ampkα rabbit monoclonal antibody, by Cell Signaling Technology (2 mentions)
Pampkα thr172 rabbit monoclonal antibody, by Cell Signaling Technology (2 mentions)
Secondary antibody against rabbit or mouse igg, by Promega (2 mentions)
Anti sept9 rabbit antibody, by Proteintech (2 mentions)
Mmp3 rabbit antibody, by Arigo Biolaboratories (2 mentions)
Cd9 mouse antibody, by Proteintech (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Chemiluminescence system, by GE Healthcare (1 mentions)
Protease inhibitor cocktail 2, by Roche (1 mentions)
Anti β actin peroxidase, by Merck Group (1 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl hrp conjugated anti mouse or anti rabbit igg, by GE Healthcare (1 mentions)
11 protocols using α tubulin mouse antibody
1
Hippocampal Neuron Lysate Preparation
2
Antibody Staining for Malaria Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All molecular biology reagents and oligonucleotides were purchased from MilliporeSigma, USA, unless otherwise stated. All oligonucleotides were purchased from Integrated DNA Technologies, Inc. (IDT), USA. The following primary antibodies and antisera and dilutions were used: mouse α-P230p (1:100, kindly gifted by Kim Williamson, Uniformed Services University of the Health Sciences, USA) α-Pfg377 (1:500, kindly gifted by Pietro Alano at Istituto Superiore di Sanità, Italy), mouse α-tubulin antibody (1:200, Sigma-Aldrich, catalog [cat.] no. T5168), rat α-mCherry antibody (1:200, Thermo Scientific cat. no. M11217, clone 16D7), α-NUP116 (1:100, rabbit, kindly gifted by Artur Scherf at Institut Pasteur, France). The following reagents were obtained through BEI Resources, NIAID, NIH: hybridoma 4B7 anti-Pfs25-kilodalton gamete surface protein (Pfs25), MRA-315, contributed by Louis H. Miller and Allan Saul and α-Pfs25 (1:1, mouse).
3
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was separated on 8% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk and incubated with rabbit anti-ITGA3 antibody (Abcam, Cambridge, UK, 1:1000 dilution) and mouse α-tubulin antibody (1:1000; Sigma-Aldrich, Ronkonkoma, NY, USA) was used as a loading control. The proteins were detected by enhanced chemiluminescence reagents.
4
Profiling Microtubule and Sarcomere Structure
5
Profiling Microtubule and Sarcomere Structure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microtubule and sarcomere structure were profiled in cells fixed in paraformaldehyde (4%) for 15 min, followed by 10 min permeabilization with Triton X-100 (0.1%). SuperBlock™ phosphate-buffered saline (PBS) was used for blocking procedure and antibody dilution (Thermo Fisher Scientific, Waltham, MA, USA). The cells were incubated overnight (4°C) with mouse α-tubulin antibody (Millipore Sigma, St Louis, MO, USA) and phalloidin conjugated with Alexa Fluor 633 (Thermo Fisher Scientific), followed by a 2 hour incubation in secondary anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific). VCMs were imaged using a Nikon A1R inverted confocal microscope with a ×60/1.4 NA oil-immersion objective (Nikon, Melville, NY, USA).
The free and polymerized tubulin fractions were isolated from snap frozen hearts as in Belanto et al. (2016) (link), with western blots for α-tubulin and its detyrosinated form (i.e., glu-tubulin, the enzymatic removal of the C-terminal tyrosine yielding a terminal glutamate), as described (Kerr et al. 2015 (link)).
The free and polymerized tubulin fractions were isolated from snap frozen hearts as in Belanto et al. (2016) (link), with western blots for α-tubulin and its detyrosinated form (i.e., glu-tubulin, the enzymatic removal of the C-terminal tyrosine yielding a terminal glutamate), as described (Kerr et al. 2015 (link)).
6
AMPK and IKK-β Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hypothalamus was homogenized in the lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 12% glycerol, 0.5 mM DTT, 0.1 mM EGTA) with a cocktail of protease inhibitors (Sigma Aldrich). Proteins (20 or 40 μg/lane) were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with AMPKα rabbit monoclonal antibody (Cell Signaling Technology; dil 1:1000), pAMPKα (Thr172) rabbit monoclonal antibody (Cell Signaling Technology; dil 1:1000), IKK-β rabbit monoclonal antibody (Abcam; dil 1:500) or α-tubulin mouse antibody (Sigma Aldrich; dil 1:1000) overnight at 4°C and then with secondary antibody against rabbit or mouse IgG (Promega; dil 1:2500) for 1 h at RT. The signals were visualized with the ECL system (Pierce). The expression level of α-tubulin was used to normalize the data.
7
Western Blot Primary and Secondary Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used for Western blots: GFP rabbit antibody (1:1000, #ab290, Abcam), CCDC170 rabbit antibody (1:500, #Ab97814, Abcam), RCAS1 rabbit antibody (1:100, #12290, Cell Signaling), acetylated α-tubulin mouse antibody (1:500, #sc-23950, Santa Cruz), α-tubulin mouse antibody (1:2000, #T5168, Sigma-Aldrich), and β-actin mouse monoclonal antibody (1:5000, Cat#A5316, Sigma-Aldrich). For secondary antibodies, ECL HRP-conjugated anti-mouse or anti-rabbit IgG (1:2000, GE Healthcare) were used.
8
Western Blot Analysis of Cell Lysates
9
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared by washing cells in cold PBS followed by addition of SDS-PAGE sample buffer containing Protease Inhibitor Cocktail (SIGMA Cat#P2714–1BTL). Cells were transferred to a 1.5ml tube, sonicated, and heated at 95°C for 5 min. Proteins were separated by electrophoresis in NuPAGE 10% Bis-Tris gels and transferred to PVDF membranes (Milipore- Immobilon, FL, Cat #IPFL00010- pore size: 0.45μm) by electroblotting. Membranes were blocked in PBS+ 5% milk powder and incubated with primary antibodies and anti-SEPT9 rabbit antibody (Proteintech Cat#10769–1-AP) and α-TUBULIN mouse antibody (Sigma Cat #T9026) overnight at 4°C. The other antibodies used were MMP3 rabbit antibody (Arigo Biolaboratories Cat#ARG55262), CD9 mouse antibody (Proteintech Cat#60232–1-Ig), or Actin mouse antibody (Proteintech Cat#66009–1-Ig). After three washes in PBS-T, membranes were incubated with secondary antibodies (Mouse 680 and Rabbit 800 [LI-COR Biosciences]) for one hour at room temperature, washed three times in PBS-T, and scanned using a high-sensitivity Odyssey Infrared Imaging System (Li-COR Biosciences). α-TUBULIN was used as a loading control. ImageJ was used for quantification of bands intensities.
10
Western Blot Analysis of AMPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hypothalamus was homogenized in the lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 12% glycerol, 0.5 mM DTT, 0.1 mM EGTA) with a cocktail of protease inhibitors (Sigma-Aldrich). Separation of proteins and Western blot analyses were performed as previously described (Chun et al., 2004 (link)). Briefly, proteins (20 or 40 μg/lane) were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with AMPKα rabbit monoclonal antibody (Cell Signaling Technology; 1:1000), pAMPKα (Thr172) rabbit monoclonal antibody (Cell Signaling Technology; 1:1000), and α-tubulin mouse antibody (Sigma-Aldrich; 1:1000) overnight at 4°C and then with secondary antibody against rabbit or mouse IgG (Promega; 1:2500) for 1 h at RT. The signals were visualized with the ECL system (Pierce). The expression level of α-tubulin was used to normalize the data.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!