Proteins were extracted using RIPA buffer from the cells, and the protein concentrations were quantified by Bio-Rad DC™ Protein Assay (Bio-Rad). Western blot was performed as described.6 (link) Briefly, protein extracts were loaded and separated in 4% stacking and 9% resolving SDS-PAGE gel. Proteins were transferred to a PVDF membrane. Proteins were probed with primary antibodies mEM48 at 1:75 dilution (courtesy of Xiao-Jiang Li), 1C2 at 1:1000 dilution (MAB1574, Chemicon), α-Tubulin at 1:1000 dilution (T9026, Sigma), and peroxidase anti-mouse IgG at 1:10,000 (715-035-150, Jackson ImmunoResearch). All blots were derived from the same experiment were processed in parallel.
Mab1574
Manufactured by Merck Group
Sourced in United States
MAB1574 is a laboratory equipment product manufactured by Merck Group. It is a monoclonal antibody designed for specific applications in research and development. The core function of MAB1574 is to provide a reliable and sensitive tool for targeted analyte detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Mab2166, by Merck Group (5 mentions)
Mab5360, by Merck Group (3 mentions)
Mn1020, by Merck Group (2 mentions)
Ab181238, by Novus Biologicals (2 mentions)
Ab1903, by Thermo Fisher Scientific (2 mentions)
Hpa035152, by Bioss Antibodies (2 mentions)
Bs 11184r, by Abcam (2 mentions)
Nb300 203, by Novus Biologicals (2 mentions)
Gapdh, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
T9026, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Fitc conjugated anti mouse antibody, by Merck Group (1 mentions)
Cellulose acetate membrane, by Sartorius (1 mentions)
Thioflavin t, by Merck Group (1 mentions)
Harmine, by Merck Group (1 mentions)
Oligomer specific a11 polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Cm3050, by Leica (1 mentions)
23 protocols using mab1574
1
Protein Extraction and Western Blot Analysis
2
Investigating MOBP and HIP1 in MSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ten cases were utilized to investigate tissue expression patterns of MOBP and HIP1. These included MSA cases (N = 5) and healthy controls (N = 5). Eight‐micrometre‐thick sections were cut from the cerebellar hemispheric FFPE blocks of patients with MSA and healthy controls and immunostained using a standard avidin‐biotin‐peroxidase complex method with di‐aminobenzidine as the chromogen. The antibodies used in this study were MOBP (Atlas Antibodies HPA035152, 1:200; Bioss Antibodies BS‐11184R, 1:100) and HIP1 (Abcam ab181238, 1:100; Novus Biologicals NB300‐203, 1:2000). Heat antigen retrieval pre‐treatment was used prior to application of the primary antibodies unless otherwise specified. The samples were mounted and examined using a light microscope. Additionally, three cases per disease control group (PD N = 3, PSP N = 3, and HD N = 3) were used to investigate the disease specificity of expression patterns of MOBP and HIP1. Mid brain was investigated in PD, and the frontal cortex in PSP and HD. FFPE tissue sections were processed and stained as described above for the cerebellar samples. FFPE tissue sections were also stained with mouse anti‐SNCA (Abcam ab1903, 1:1000), mouse anti‐AT8 (Invitrogen MN1020, 1:600) and mouse anti‐IC2 (Chemicon MAB1574, 1:1000, formic acid pre‐treatment) for PD midbrain, and frontal cortex for PSP and HD respectively.
3
Yeast Prion Protein Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
S. cerevisiae BY4742 [MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0, (RNQ1+)] is a product of Open
BioSystems and was purchased from SAF Labs Pvt. Ltd. Mumbai, India.
Harmine, glutathione-agarose matrix, thioflavin T, dihydroethidium
(DHE), and the antimouse FITC conjugated antibody were purchased from
Sigma-Aldrich, Bengaluru, India. Mouse anti-polyglutamine (polyglutamine
expansion disease marker monoclonal antibody, MAB1574) was a product
of Chemicon International and was purchased from Millipore (India)
Pvt. Ltd., New Delhi, India. A goat anti-Rnq1 antibody was purchased
from Santa Cruz, California, USA. The oligomer-specific A11 polyclonal
antibody was purchased from Invitrogen Corporation, California, USA.
The nitrocellulose membrane (0.2 μm) was purchased from Advanced
Microdevices Pvt. Ltd. Ambala Cantt, India. The cellulose acetate
membrane was purchased from Sartorius Stedium Biotech, Goettingen,
Germany. All other reagents and chemicals used were of analytical
grade or higher.
BioSystems and was purchased from SAF Labs Pvt. Ltd. Mumbai, India.
Harmine, glutathione-agarose matrix, thioflavin T, dihydroethidium
(DHE), and the antimouse FITC conjugated antibody were purchased from
Sigma-Aldrich, Bengaluru, India. Mouse anti-polyglutamine (polyglutamine
expansion disease marker monoclonal antibody, MAB1574) was a product
of Chemicon International and was purchased from Millipore (India)
Pvt. Ltd., New Delhi, India. A goat anti-Rnq1 antibody was purchased
from Santa Cruz, California, USA. The oligomer-specific A11 polyclonal
antibody was purchased from Invitrogen Corporation, California, USA.
The nitrocellulose membrane (0.2 μm) was purchased from Advanced
Microdevices Pvt. Ltd. Ambala Cantt, India. The cellulose acetate
membrane was purchased from Sartorius Stedium Biotech, Goettingen,
Germany. All other reagents and chemicals used were of analytical
grade or higher.
4
Cerebellar Protein Expression in MSA
5
Immunofluorescence Analysis of Cerebellar Purkinje Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were euthanized in a CO2 chamber, and their whole brains were perfused and fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). The brain samples were cut into serial 25 μm sections using a cryostat microtome (Leica CM3050S, Wetzlar, Germany). For immunofluorescence staining, cerebellar tissue sections were blocked with 1% bovine serum albumin (BSA) blocking solution for 60 min. Permeabilized samples were probed using appropriate dilution of antibodies: anti-spinocerebellar ataxia type 3 (MAB5360, Merck Millipore, Billerica, MA, USA), anti-polyglutamine-expansion disease marker (MAB1574, Merck Millipore), and anti-calbindin (ab108404, Abcam, Cambridge, UK) in blocking solution for overnight within a humidified chamber at 4 °C. Tissue slides were then washed three times in PBS for 10 min each and incubated with fluorophore-conjugated (Alexa Fluor 594 or Alexa Fluor 488 dyes, Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies in blocking solution for 60 min at room temperature. Samples were also labeled with Hoechst 33342 to mark the nuclei, mounted with anti-photobleaching medium, and observed using confocal microscopy (Zeiss LSM510, Oberkochen, Germany). A 20× or 40× oil immersion objective was used to capture Z stack images of Purkinje cell layer (25 μm thickness), and ZEN Blue software was applied to export the images.
6
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 50 μg of protein was diluted in 1% (v/v) Laemmli buffer and heated at 60°C for 5 min, followed by separation on a 6% SDS-polyacrylamide gel for 3 h at 110 V and subsequent overnight transfer on a low fluorescence polyvinylidene difluoride (PVDF) membrane (Immobilon-FL Merck-Millipore no. IPFL00010) at 20 V in transfer buffer containing 0.01% (w/v) SDS and 16% (v/v) methanol. REVERT total protein stain was performed as described above. The membranes were then blocked with 2.5% (w/v) BSA prepared in tris-buffered saline (TBS) and sequentially incubated overnight at 4°C with anti-HTT (1:1,000, Merck-Millipore, no. MAB2166) and anti-polyglutamine expansion (1:500, Merck-Millipore, no. MAB1574) primary antibodies diluted in TBS containing 0.1% (v/v) Tween 20 (TBST). The membranes were washed three times and incubated with IRDye 800CW (LI-COR Biosciences, no. 926-32212) secondary antibody for 45 min at RT. Membrane revelation and quantification were performed as detailed above.
7
Quantification of Soluble Mutant Ataxin-3 by TR-FRET
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Soluble mutant ataxin‐3 measurements via time‐resolved Förster resonance energy transfer immunoassay (TR‐FRET) were performed using a combination of an anti‐ataxin‐3 antibody (clone 1H9, MAB5360, Merck) labelled with Tb (donor) fluorophore and an anti‐polyQ antibody (clone 1C2, MAB1574, Merck) labelled with d2 (acceptor) fluorophore (labelling by Cisbio). Briefly, homogenised cerebellar and frontal lobe samples for Nf measurements were diluted in homogenisation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, cOmplete Protease Inhibitor Cocktail) to a final concentration of 1 μg/μl total protein amount. Next, 5 μl of diluted sample was incubated with 1 μl of the TR‐FRET antibody mix (1 ng 1H9‐Tb + 3 ng MW1‐d2 in 50 mM NaH2PO4, 400 mM NaF, 0.1% BSA, 0.05% Tween‐20) in a low‐volume white ProxiPlate 384 TC Plus plate (PerkinElmer) at 4°C for 22 h. Signals were detected at 620 nm and 665 nm using an EnVision Multimode Plate Reader with a TRF‐laser unit (PerkinElmer).
8
Quantitative Polyglutamine Aggregation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polyglutamine aggregates were detected with a modified filter assay (Scherzinger et al., 1997 (link)). In brief, 25 heads were homogenized in 200 µl Cytoplasmic Extraction Reagent I buffer and fractionated using NE-PER Nuclear and Cytoplasmic Extraction Reagents following the manufacturer’s protocol (Thermo Fisher Scientific). Cytosolic fractions were adjusted to 1% SDS, incubated for 15 min at room temperature, denatured at 95°C for 5 min, and filtered through a 0.2-µm cellulose acetate membrane (Sterlitech Corporation) preequilibrated with 1% SDS. Filters were washed twice with 0.2% SDS and blocked in TBS (100 mM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 3% nonfat dried milk before development with mouse 1C2 antibody (1:1,000; MAB1574; EMD Millipore) and detection and quantification with Odyssey scanner and software.
9
Antibody Identification of Huntingtin Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used to identify Htt (see Supplementary Fig. S1B for the epitope recognized): monoclonal mouse anti-HTT (1:1000 for immunoblotting and 1:500 for immunofluorescence, cod. MAB2166, EMD Millipore Corporation, Billerica, MA, USA); monoclonal mouse anti-HTT1C2 Ms X Polyglutamine (1:1000 for immunoblotting, cod. MAB1574, EMD Millipore Corporation). Other antibodies used were: rabbit anti-SQTM1/p62 (1:500, cod. 8025, Cell Signaling Technology, Danvers, MA U.S.A.), rabbit anti-LC3B (1:1000, cod. L7543, Sigma Aldrich Corp.), rabbit anti-Cathepsin D (1:100 for immunofluorescence, produced in our laboratory (Erdmann et al., 2007) ; mouse anti-LAMP1 (1:1000 for immunofluorescence, cod. 555798, Becton, Dickinson and Company, New Jersey, NJ, USA), mouse anti-btubulin (1:1000 for immunoblotting, cod. T5201, Sigma Aldrich Corp.), mouse anti-p21 (1:100, cod. sc-817, Santa Cruz Biotechnology Inc. Dallas, TX, USA), polyclonal rabbit anti-MKI67 (Ki-67, 1:100 for immunostaining, cod. HPA001164, ATLAS-Sigma Prestige Antibodies, Sigma-Aldrich Corp.), mouse anti-GFP (1:1000, cod. 632381, Living Color Monoclonal Antibody (JL-8), Clontech Laboratories Inc. Mountain View, CA U.S.A.).
10
Protein Immunodetection in Neural Tissues
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!