Western blotting was performed according to standard protocols (BioRad). Whole cell lysates were obtained from snap-frozen cell pellets using the cOmplete™ Lysis-M EDTA-free lysis buffer (Roche). Protein concentration was determined by Pierce BCA assay (ThermoFisher Scientific) and used to maximise equal loading across the gels (~9 μg per lane). Electrophoresis was run on 4–12% Criterion™ XT Bis-Tris Protein Gel (BioRad) at constant voltage (200 V, one hour) and followed by protein transfer to a nitrocellulose membrane (BioRad). Blocking was performed in PBS, 0.1% Tween, 5% dry milk powder (Marvel) at RT for one hour followed by primary antibody incubation overnight at 4 °C. Primary antibodies were diluted in PBS, 0.1% Tween, 5% dry milk powder as follows: mouse anti SFPQ (Abcam, ab11825) 1:250; rabbit anti TLS/FUS (Abcam, ab84078) 1:500; rabbit anti DDX39 (Sigma Aldrich, SAB2700315) 1:500, rabbit anti DARS (Abcam, ab182157) 1:1000; mouse anti GLN1 (Antibodies-online, AA 1-373) 1:1000; rabbit anti TDP-43 (Abcam, Ab133547) 1:10,000; mouse anti GAPDH (Life Technologies, clone 6C5, AM43000) 1:5000. For detection membranes were incubated with species-specific near infra-red fluorescent antibodies (IRDye, Licor) for one hour at RT and imaged using an Odyssey Fc Imaging System (Licor).
Ab11825
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab11825 is a recombinant monoclonal antibody that recognizes Human CD79A. The antibody is designed for use in various immunoassays to detect the presence and quantity of CD79A in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Criterion xt bis tris protein gel, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Complete lysis m edta free lysis buffer, by Roche (1 mentions)
Ab1162, by Abcam (1 mentions)
Ab184305, by Abcam (1 mentions)
Ab70335, by Abcam (1 mentions)
Ab40793, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
Ab207321, by Abcam (1 mentions)
Rabbit anti eif2α, by Cell Signaling Technology (1 mentions)
Nb600 241, by Novus Biologicals (1 mentions)
Protein a bead, by Thermo Fisher Scientific (1 mentions)
Dynal magnetic bead, by Thermo Fisher Scientific (1 mentions)
Ab171870, by Abcam (1 mentions)
Ab54708, by Abcam (1 mentions)
Ab54593, by Abcam (1 mentions)
Ab5832, by Abcam (1 mentions)
8 protocols using ab11825
1
Western Blot Analysis of Protein Targets
2
Protein Expression Analysis in DEN-Treated Cells
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SMMC-7721 or MHCC97h cells were treated with DEN (4 mM) or not, lysed, and then subjected to SDS-PAGE and immunoblotting, as previously described [24 (link)]. Primary antibodies against NONO (1:500; ab70335, Abcam, USA), ACLY (1:500; ab40793, Abcam, USA), Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) (1:500; ab184305, Abcam, USA), Splicing factor proline and glutamine rich (SFPQ) (1:500; ab11825, Abcam, USA), Flag (1:1000; ab1162, Abcam, USA), Myc (1:1000; ab32072, Abcam, USA), and GAPDH (1:1000; ab181602, Abcam, USA) were used.
3
Quantitative Protein Expression Analysis
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Diluted protein lysate was mixed with fluorescent master mix and heated at 95 °C for 5 min. Three μL of protein mix (1 mg/mL maximal concentration) containing Protein Normalization Reagent, blocking reagent, wash buffer, target primary antibody (rabbit anti-eIF2α [Cell Signaling Technology 9721]) diluted 1:50, mouse anti-phospho-eIF2α [Cell Signaling Technology 2103] diluted 1:50, mouse anti-p21 antibody [Santacruz, sc-6246] diluted 1:50, rabbit anti-P54nrb diluted 1:200 [Santacruz, sc-67016], rabbit anti-PSPC1 diluted 1:100 [bethyl laboratory, A303-205A], mouse anti-SFPQ diluted 1:100 [Abcam, Ab11825]; rabbit anti-FGF1 diluted 1:25 [Abcam Ab207321], rabbit anti-Nucleolin diluted 1:50 [Novus biological, NB600-241], secondary-HRP (ready to use rabbit or mouse ‘detection module’, DM-001 or αDM-002), and chemiluminescent substrate were dispensed into designated wells in a manufacturer-provided microplate. The plate was loaded into the instrument (Jess, Protein Simple) and proteins were drawn into individual capillaries on a 25 capillary cassette (12–230 kDa) (SM-SW004). Normalization reagent allow detecting total protein in the capillary through the binding of amine group by a biomolecule and to get rid of housekeeping protein that can arbor an inconsistent and unreliable expression. Graph plotted in Figures represent chemiluminescence value before normalization.
4
ChIP-qPCR Analysis of PSF Binding
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Cells were cultured were seeded in 10-cm dishes to form an 80% confluent monolayer. For each sample, 1×107 cells were harvested. ChIP for PSF was conducted using a murine monoclonal antibody against PSF (cat. no. ab11825, Abcam, Cambridge, UK) or a non-specific rabbit IgG (control, cat. no. ab171870, Abcam) conjugated to secondary Dynal magnetic beads (Thermo Fisher Scientific, Inc.). Cells were cross-linked with 1% formaldehyde for 15 min at room temperature and sonicated at 30% for 10 sec and left on ice for 1 min. The sonication step was repeated 10 times. Next, 10 µg of either antibody or control was added into 100 µl lysate for a 4-h incubation at 4°C with 10 µl protein-A beads (Thermo Fisher Scientific, Inc.). The beads were washed three times with washing buffer (150 mM NaCl, 5 mM MgCl2, 10 mM HEPES, pH 7.0, 1% NP-40) and eluted in 300 µl 3 M NaCl elution buffer for 3 h at 65°C. The ChIP products were analyzed by RT-qPCR following the aforementioned protocol. Dihydrofolate Reductase (DHFR) 5′ untranslated region (UTR) was used as a negative control sequence. The primers for detecting the PSF-reacting region on the GAGE6 promoter were as follows: 5′-GCCTTCTGCAAAGAAGTCTTGCGC-3′ (forward) and 5′-ATGCGAATTCGAGGCTGAGGCAGACAAT-3′ (reverse); DHFR 5′UTR, 5′-CTGATGTCCAGGAGGAGAAAGG-3′ (forward) and 5′-AGCCCGACAATGTCAAGGACTG-3′ (reverse).
5
Quantification of Arginine Methylation
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TCR-activated PBMCs employed for the MethylScan study were used to generate lysates as described above for Western blot analysis. Protein concentrations were determined using the BCA Protein Assay Kit. The anti-ADMA antibody (CST, clone D10F7A10) was biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific, #21326) according to the manufacturer’s instructions. 1 µg of lysates, in triplicates, was incubated with the biotinylated anti-ADMA antibody, at a concentration of 0.5 µg/mL, and antibodies specific to ALYREF (Abcam, #ab6141), DHX9 (Abcam, #ab54593), EIF4H (Abcam, #ab77455), EWSR1 (Abcam, #ab54708), G3BP1 (Abcam, #ab56574), HNRNPA1 (Abcam, #ab5832), KHDRSB1 (Abcam, #ab56836), SFPQ (Abcam, #ab11825) and TPR (Abcam, #ab58344) at a concentration of 1 µg/mL. After a 45-min incubation at room temperature, streptavidin-coated acceptor beads (Perkin Elmer, #AL125C) and anti-mouse IgG-coated donor beads (Perkin Elmer, #AS104) were added to respective wells of 96 well microplates (Corning, #3693), at a final concentration of 20 µg/mL and incubated for an additional 45 min at room temperature. All solutions were prepared in AlphaLISA universal buffer (Perkin Elmer, #AL001F) and microplates were shaken after each addition and centrifuged prior to luminescence detection on an Envision microplate reader (Perkin Elmer).
6
Immunolabeling of Neural Cell Markers
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For immunocytochemistry and immunohistochemistry, samples were blocked in 10% normal goat serum (NGS) or 10% normal donkey serum (NDS) as appropriate and permeabilised in 0.3% Triton X-100 (Sigma-Aldrich; in PBS) at room temperature (RT) for 1 h. Immunolabelling was performed with primary antibodies in NGS (5%) and Triton X-100 (0.1% in PBS) at 4 °C overnight followed by species-specific secondary antibodies for 1 h at RT and DAPI nuclear counterstain (100 ng/ml) for 10 min at RT. For human post-mortem samples fixation and permeabilisation in cold methanol (−20 °C, 20 min) was performed before the immunostaining. Primary antibodies were diluted as follows: rabbit anti Olig2 (Millipore, AB9610) 1:200; mouse anti SMI32 (Cambridge Bioscience, SMI-32R-500) 1:1000; goat anti ChAT (Millipore, AB144P) 1:100; rabbit anti Pax6 (biolegend, 901301) 1:300; mouse and rabbit anti SFPQ (Abcam, ab11825 and ab38148, respectively) 1:100; mouse and rabbit anti beta-tubulinIII (biolegend, 801201), mouse anti Lim3 (DSHB, 67.4E12) 1:50; chicken anti Nkx2.2 (DSHB, 74.5A5) 1:50. Images were acquired using either a 710 Laser Scanning Confocal Microscope (Zeiss) or the Opera Phenix High-Content Screening System (Perkin Elmer). For image analysis, Fiji or the Columbus Image Analysis System (Perkin Elmer) were used.
7
Visualizing NEAT1 RNA and PSPC1/SFPQ Proteins in HEK293T Cells
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HEK293T cells were grown on a glass slide and fixed in 4% formaldehyde in PBS. Cells were washed once with PBS, permeabilized with Triton X-100, dehydrated through a series of ethanol washes and hybridized overnight with a digoxigenin-labeled NEAT1 probe (Table S1). RNA FISH signal was detected by incubating with Alexa Flour 647-labeled anti-digoxygenin antibody (Roche) and examined on a Leica TCS SP8 STED microscope.
Immunofluorescence was carried out as described previously (Deng et al., 2019 (link)). Briefly, cells growing on glass slides were rinsed with PBS and fixed with 4% PFA for 15 min, and then permeabilized in 0.2% Triton X-100 followed by 30 min 5% BSA blocking. The cells were then stained with human anti-PSPC1 (Abcam, ab104238) or anti-SFPQ antibodies (Abcam, ab11825) in blocking buffer for overnight at 4 °C and washed three times with PBS. Then stained with Alexa Flour 647-labeled secondary antibodies (Jackson Laboratory) in blocking buffer for 1 h. After three washes with PBS, coverslips were mounted with VECTASHIELD mounting medium containing 0.5 μg/mL DAPI and then visualized at 100× on a CCD camera mounted on a Leica TCS SP8 STED microscope using imaging software. The magenta signal was defined as green by ImageJ (National Institutes of Health) before further colocalization analysis.
Immunofluorescence was carried out as described previously (Deng et al., 2019 (link)). Briefly, cells growing on glass slides were rinsed with PBS and fixed with 4% PFA for 15 min, and then permeabilized in 0.2% Triton X-100 followed by 30 min 5% BSA blocking. The cells were then stained with human anti-PSPC1 (Abcam, ab104238) or anti-SFPQ antibodies (Abcam, ab11825) in blocking buffer for overnight at 4 °C and washed three times with PBS. Then stained with Alexa Flour 647-labeled secondary antibodies (Jackson Laboratory) in blocking buffer for 1 h. After three washes with PBS, coverslips were mounted with VECTASHIELD mounting medium containing 0.5 μg/mL DAPI and then visualized at 100× on a CCD camera mounted on a Leica TCS SP8 STED microscope using imaging software. The magenta signal was defined as green by ImageJ (National Institutes of Health) before further colocalization analysis.
8
Investigating ARAP1-AS1 Protein Interactions
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ARAP1‐AS1 and anti‐sense biotin‐labeled probes were designed and synthesized (RiboBio) and subsequently incubated with protein lysates of CaSki and SiHa cells at 4°C overnight on a rotator. Next, the pierce streptavidin magnetic dynabeads (Invitrogen) were added into above protien‐probe complex and incubated for another two hours at room temperature. Lastly, the protein was eluted and analyzed by western blot assay. Western blot assay was performed in accordance with routine protocol, including electrophoresis, transfer, blocking, incubation with corresponding antibodies and final exposure. The primary antibodies used in this study were as follows: anti‐PSF (ab11825, Abcam), anti‐PTB (ab5642, Abcam), anti‐MYC (ab56, Abcam) and anti‐GAPDH (ab9485, Abcam). Besides, RIP assay was conducted out using Imprint® RIP kit (Sigma‐Aldrich) according to manufacturer's instruction.
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