Western blotting was used for comparison of expression levels of CA-VI and cystatins. Samples from the individuals run in SDS PAGE profiles were analysed in triplicate. After protein separation by SDS PAGE (5 μg total protein from each sample), in 14% polyacrylamide gels (100 V constant voltage) (mini-protean apparatus, Bio-Rad), proteins were transferred to a PDVF membrane by electroblotting using a Tris-glycine buffer system. After transferring, blocking was performed with 5% non-fat dry milk in TBS-Tween 20, for 2 h, with agitation, at room temperature. The membrane was cut, with the upper part incubated with primary antibody anti-CA-VI (Santa Cruz Biotechnology sc-99173; dilution: 1:200) and the lower part with primary antibody anti-cystatin S-SA-SN (Santa Cruz Biotechnology sc-73884; dilution: 1:200), overnight at 4°C. CA-VI and cystatin bands were detected with an alkaline phosphatase-linked secondary antibody (anti-rabbit and anti-mouse, respectively, GE Healthcare, 1:10,000 dilution), using a chemifluorescent substrate (ECF Plus Western Blotting Detection Reagents, GE, Healthcare). Membranes were revealed in a transilluminator (Gel-Doc, Bio-Rad) and a semi-quantitative analysis of band expression was carried out using the software Bio-Rad Image Lab 5.2.1.
Anti mouse and anti rabbit
Manufactured by GE Healthcare
Sourced in United Kingdom
Anti-mouse and anti-rabbit are secondary antibodies used in various laboratory techniques, such as Western blotting, immunohistochemistry, and ELISA. These antibodies are designed to specifically bind to primary antibodies that have been raised against mouse or rabbit proteins, allowing for the detection and visualization of target analytes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti goat, by Santa Cruz Biotechnology (3 mentions)
β actin, by Merck Group (3 mentions)
Rpn2232, by GE Healthcare (2 mentions)
Na931v, by Santa Cruz Biotechnology (2 mentions)
Na934v, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ecf plus western blotting detection reagents, by GE Healthcare (1 mentions)
Mini protean apparatus, by Bio-Rad (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Gel doc, by Bio-Rad (1 mentions)
Fer 5d2, by Cell Signaling Technology (1 mentions)
Histone h3 d1h2 xp, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Py100, by Cell Signaling Technology (1 mentions)
Met d1c2 xp, by Cell Signaling Technology (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
Odyssey fc imager, by LI COR (1 mentions)
Actin, by Merck Group (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Anti puromycin, by Kerafast (1 mentions)
12 protocols using anti mouse and anti rabbit
1
Quantifying Salivary CA-VI and Cystatins
2
Immunoblotting Techniques for Protein Profiling
3
Western Blot Analysis of Signaling Proteins
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All tissues were dissociated in RIPA buffer (unless otherwise specified). Western blotting was previously described (Gkogkas et al. 2013 (link)). Antibodies against indicated proteins were: eIF4E, phospho-eIF4E (Ser209) (BD Biotechnologies), rpS6, phospho-rpS6(240–244) (Cell Signaling), anti-puromycin (Kerafast) and β-tubulin (SIGMA). Secondaries were anti-mouse and anti-rabbit (GE Healthcare), and anti-goat (Santa-Cruz) antibodies. Quantification of immunoblots was performed using ImageJ (NIH). Values were normalized to β-tubulin or another control where specified, and presented as a ratio of phosphoprotein/total protein.
4
Antibody Immunoblotting Protocol
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Anti-USP14 (Cell Signaling Technology, #11931), anti-β-actin (MBL, 177-3), anti-HA-tag (MBL, 561), anti-LC3B (Cell Signaling Technology, #2775), anti-p53 (Cell Signaling Technology, #2524), and anti-PrP (Santa Cruz Biotechnology, M20; SPI-Bio, SAF32 and SAF83) antibodies were purchased from the indicated vendors. The anti-PrP mAb132 was a kind gift from Prof. Motohiro Horiuchi (Hokkaido University). Horseradish peroxidase-conjugated anti-goat (Jackson ImmunoResearch), anti-mouse, and anti-rabbit (GE Healthcare Life Sciences) IgG antibodies were used for immunoblotting.
5
MTT Assay and Pharmacological Targets
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(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT and L-glutamine were obtained from USB (Cleveland, OH, USA). Mouse mAb anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The PGE2 enzyme immunoassay kit, Valeryl Salicylate, compounds NS-398, AH6809, AH23848, SC-19220, L-798106 and polyclonal antibodies against COX-1, COX-2, mPGES-1 and the EP4 receptor were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Pyrrolidine-2 (Pyr-2) was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). Secondary antibodies, anti-mouse and anti-rabbit, conjugated to HRP and nitrocellulose membrane, were obtained from GE Healthcare (Buckinghamshire, UK). The Cytometric Bead Assay (CBA) kit was purchased from BD Bioscience (San Jose, CA, USA). Gentamicin was purchased from Schering-Plough (Whitehouse Station, NJ, USA), DMSO from Amresco (Solon, OH, USA) and Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum and real-time polymerase chain reaction (PCR) assay kit from Life Technologies (São Paulo, SP, Brazil).
6
Western Blot Analysis of RZZ Complex
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Samples were diluted in Laemmli sample buffer, boiled at 95°C, resolved on SDS-PAGE with NuPAGE Bis-Tris 4–12% gradient gels (Invitrogen), and transferred onto nitrocellulose membranes (GE Healthcare). The following primary antibodies were used: anti-ROD (mouse monoclonal; clone CB22-1; 1:500), anti-Zwilch (mouse monoclonal; clone CE47-3; 1:500), anti-ZW10 (mouse monoclonal; clone CO-45-2; 1:500), and anti-Spindly (rabbit polyclonal; A301-354A; 1:5,000; Bethyl Laboratories, Inc.). Mice immunized with recombinant full-length RZZ complex were used to generate anti-ROD, anti-ZW10, and anti-Zwilch antibodies. Antibodies were then affinity purified from sera by using immobilized antigens. Antibody productions, purifications, and biochemistry were performed at the Istituto FIRC di Oncologia Molecolare–Instituto Europeo de Oncologia (IFOM-IEO) Campus Biochemistry Unit. Secondary antibodies were anti–mouse and anti–rabbit (working dilutions, 1:10,000; GE Healthcare). After incubation with the ECL Western blotting system (GE Healthcare), images were acquired with the ChemiDoc MP Imaging System (Bio-Rad Laboratories) in 8-bit Tiff format.
7
Immunoblotting Protocol with Antibody Detection
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Immunoblots were performed as described previously.75 (link) The primary antibodies used are presented in Table S7. Membranes were analyzed using the appropriate peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit from GE Healthcare UK Limited, NA931V and NA934V, and anti-goat from Santa Cruz Biotech, sc-2020). Proteins were detected by enhanced chemiluminescence (GE Healthcare, RPN2232).
8
Comprehensive Immunostaining Protocol
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Primary antibodies: HT7 (mouse, Thermo Scientific, 1:1,000), AT8 (mouse, Thermo Scientific, 1:1,000), NF200 (rabbit, Sigma–Aldrich, 1:1,000), APP (clone 22C11, mouse, Millipore, 1:1,000), Iba1 (rabbit, Wako, 1:5,000), CD11b (rat, Serotec, 1:1,000), Olig2 (rabbit, Millipore, 1:1,000), NG2 (rabbit, Millipore, 1:1,000) APC (mouse, clone CC1, Chalbiochem, 1:200), SOX2 (goat, Santa Cruz, 1:300), MBP (rat, Serotec, 1:300 in IHC, 1:500 for ICC, and 1:2,000 for WB), βIII‐tubulin (Rabbit, AbCam, 1:500), β‐actin (mouse, Sigma–Aldrich, 1:10,000). Biotin‐conjugated secondary antibodies (Vector, 1:1,000), Alexa‐conjugated secondary antibodies (Molecular Probes, 1:1000), HRP‐conjugated secondary antibodies (1:5000): anti‐mouse and anti‐rabbit (GE), anti‐rat (Vector).
9
Quantitative Western Blot Analysis
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Mouse tissues were dissociated using Bio-Plex Cell Lysis Kit (Bio-Rad, Mississauga, ON, Canada). Western blotting was performed as previously described68 (link). Antibodies against indicated proteins were: phospho-eIF4E (Ser209, Abcam, 1:1000), eIF4E (BD Biosciences, 1:1000), IκBα (Cell Signaling Technology, 1:1000), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, coupled to Horseradish Peroxidase, Abcam, 1:5000) or β-actin (Sigma, 1:5000); secondary antibodies were anti-mouse and anti-rabbit (GE Healthcare). Quantification of immunoblots was performed using ImageJ (NIH), and expressed as a ratio (either p-eIF4E/eIF4E or eIF4E/GAPDH or eIF4E/β-actin, IκBα/GAPDH). Western blots experiments were replicated at least two times. Uncropped western blots are provided in Supplementary Figure 9 .
10
Western Blotting and Immunofluorescence Assay Antibodies
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The following antibodies were used for Western blotting and immunofluorescence assays: mouse anti-HA (MERCK), rabbit anti-HA (MERCK), mouse anti-Myc (Santa-Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-PINK1 (Novus Biologicals, Englewood, CO, USA), mouse anti-TIM23 (BD Biosciences, Franklin Lakes, NY, USA), mouse anti-TOM20 (BD Biosciences), mouse anti-Parkin (Santa-Cruz Biotechnology), rabbit anti-GAPDH (Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-LC3B (MERCK) and rabbit anti-cleaved PARP (Cell Signaling Technologies). HRP-conjugated secondary antibodies were as follows: anti-Mouse and anti-Rabbit (both from GE Healthcare, Little Chalfont, UK). Secondary antibodies used in immunofluorescence experiments were conjugated with either Alexa Fluor 488, Alexa Fluor 555 or Alexa Fluor 405 (Thermo Fisher Scientific).
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