Total lysates were obtained from human articular chondrocyte cultures 48 h after transfection with empty pcDNA expression vector of pcDNA expression vector containing full length AnxA6 cDNA or from late stage human OA articular cartilage using T-PERTM Tissue Protein Extraction Reagent from Pierce Biology Products. To extract the nuclear and plasma membrane proteins from the cell cultures we used the NE-PERTM Nuclear and Cytoplasmic Extraction Reagent or the Mem-PERTM Plus Membrane Protein Extraction Kit from Pierce Biology Products and followed the manufacturer’s instructions. Thirty μg of total cytoplasmic, nuclear or plasma membrane protein fractions were analyzed by SDS PAGE and immunoblotting with antibodies specific for AnxA6 (Abcam, ab7671), ß-catenin (Abcam, ab6302), Dishevelled 1 (Dvl1, Abcam, ab174679) as described previously [20 (link)]. For normalization of the protein expression levels, the membranes were immunostained with antibodies specific for beta-actin (total tissue cell lysate, Abcam, ab20272), alpha 1 sodium potassium ATPase (ATP1A1, plasma membrane fraction, Abcam, ab7671) and lamin B (nuclear fraction, Abcam, ab194109).
Ab7671
Manufactured by Abcam
Sourced in United Kingdom, United States, Germany
Ab7671 is a primary antibody produced in rabbit that recognizes the human Nucleolin protein. It is suitable for use in various immunoassay applications, including Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ez link sulfo nhs ss biotin, by Thermo Fisher Scientific (3 mentions)
Ab81321, by Abcam (2 mentions)
Ab59459, by Abcam (2 mentions)
Ab15523, by Abcam (2 mentions)
Neutravidin beads, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Gtx113390, by GeneTex (1 mentions)
Ab2826, by Abcam (1 mentions)
Ab92726, by Abcam (1 mentions)
C5922, by Merck Group (1 mentions)
Chemocam system, by Intas (1 mentions)
Mms 435p, by Fortrea (1 mentions)
Ab18956, by Abcam (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Orca r2, by Zeiss (1 mentions)
Axio observer, by Zeiss (1 mentions)
Ab167453, by Abcam (1 mentions)
43 protocols using ab7671
1
Extraction and analysis of AnxA6 in human chondrocytes
2
Immunoblot Analysis of ATPase Subunits
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Immunoblot analyses made use of the anti-sodium/potassium alpha 1 ATPase antibody (ab7671; Abcam Inc., Toronto, ON, Canada) and the anti-sodium/potassium beta 1 ATPase antibody (GTX113390; GeneTex Inc., Irvine, CA, USA). Anti-mouse (170–6516) and anti-rabbit (170–6515) horseradish peroxidase-conjugated secondary antibodies were sourced from Bio-Rad Laboratories, Inc., Hercules, CA, USA.
3
Immunoblotting Protocol for Myelin Proteins
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Immunoblotting was performed as described (Patzig et al., 2016a (link); Schardt et al., 2009 (link)). Antibodies were specific for ANLN (Acris AP16165PU-N; 1:1000), SEPT2 (ProteinTech Group 11397–1-AP; 1:500), SEPT4 (IBL JP18987; 1:500), SEPT7 (IBL JP18991; 1:5000), SEPT8 (ProteinTech Group 11769–1-AP; 1:2500), MAG ((Erb et al., 2003 (link)); kindly provided by N. Schaeren-Wiemers, Basel; 1:500), PLP/DM20 (A431 ((Jung et al., 1996 (link)); 1:5000), cyclic nucleotide phosphodiesterase (CNP) (Sigma C5922; 1:1000), MOG (clone 8–18 C5 (Linnington et al., 1984 (link)); 1:5000; kindly provided by C. Linington, Glasgow), SIRT2 (abcam 67299; 1:500), CD9 (abcam ab92726; 1:2000), CA2 ((Ghandour et al., 1980a (link); Ghandour et al., 1980b (link)); 1:1000; kindly provided by S. Ghandour, Strasbourg), ATP1A1 (abcam ab7671; 1:2500), ATP1A3 (abcam ab2826; 1:1000), beta3-Tubulin (TUBB3/Tuj1) (Covance MMS-435P; 1:1000). Secondary HRP-coupled antibodies were anti-mouse (dianova 715-035-020; 1:1000), -rabbit (dianova 711-035-152; 1:1000), or -goat (dianova 705-035-003; 1:500). Immunoblots were scanned using the Intas ChemoCam system.
4
Western Blot Analysis of NRP1, Na/K-ATPase, GDNF
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After blocking by 5% skimmed milk, the samples were incubated with primary antibody (rabbit anti-NRP1, ab81321, Abcam, 1:250; mouse anti-alpha 1 Na/K-ATPase, ab7671, Abcam, 1:250; rabbit anti-GDNF, ab18956, Abcam, 1:100; mouse anti-β-actin, sc47778, Santa cruz, 1:1000; rabbit anti-Histone H3 antibody, BS1660, bioworld, 1:500) at 4°C overnight [57 (link)]. Then, the samples were incubated with IRdye secondary antibodies (goat anti-Rabbit, LI-COR, Odyssey, 1:1000; goat anti-Mouse, LI-COR, Odyssey, 1:1000) at room temperature for 2h. Finally, the protein bands were scanned by Odyssey imaging system (LI-COR, USA) and quantified with ImageJ software (National Institutes of Health, USA).
5
Enrichment and Detection of Aquaporin-5
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The HSG cells were transiently transfected with the pEGFP-C1-AQP-5 plasmid using Lipofectamine 2000 [27 (link)]. AQP-5-transfected HSG cells were grown in a 60-mm dish, pre-incubated with MβCD for 3 min, and treated with carbachol or thapsigargin for 20 min at 37 °C. The membrane fraction was obtained as previously described [28 (link)] and sonicated in ice-cold 20 mM HEPES solution containing 1 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, and 0.3 mM phenylmethylsulfonyl fluoride (3 × 30 s; Sigma) at pH 7.4. The samples were centrifuged at 600× g at 4 °C, and the supernatants were centrifuged further at 20,000× g at 4 °C. The pellets (P2 membrane fractions) were separated via SDS-PAGE, and the proteins were transferred onto a PVDF membrane for immunoblotting. After blocking, the membrane was incubated with anti-AQP-5 (Santa Cruz, sc-9891, 1:200) as the primary antibody. The membrane was washed, incubated with donkey anti-goat IgG-HRP, and subjected to an electrochemiluminescence assay for detection. For normalization of the AQP5 signal, the membranes were stripped and re-probed with antibodies for anti-α1 Na+/K+-ATPase (Abcam, ab7671, 1:1000).
6
Immunostaining of Alveolar Type II Cells
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Alveolar epithelial cells type II were fixed with 0.1% formaldehyde for 10 min at RT, rinsed with 10% Donkey serum-PBS, incubated over-night with primary Ab (α-Na,K-ATPase, 1:100, Cat# ab7671, Abcam) at 4°C, followed by fluorescence-labeled secondary Ab incubation. Dapi was used for nuclear DNA staining. Zeiss Axio observer inverted motorized fluorescent microscope equipped with Hamamtsu Orca R2 and Zeiss HS cameras was used for microscope imaging.
7
Quantitative Immunohistochemical Scoring Protocol
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The immunohistochemical staining protocol and semiquantitative analysis were carried out following the protocol of our previous study44 (link). Primary antibodies included anti-Hsp90α (#ab59459, Abcam; Cambridge, MA), anti-ATP1A1 (#ab7671, Abcam; Cambridge, MA) and anti-STAT3 (#ab15523, Abcam; Cambridge, MA).
Following a hematoxylin counterstaining, immunostaining was scored by two independent experienced pathologists, who were blinded to the background information of the mice. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0%), 1 (1–10%), 2 (11–50%) and 3 (>50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A final immunoreactive score (IRS) was obtained for each case by multiplying the percentage and the intensity score.
Following a hematoxylin counterstaining, immunostaining was scored by two independent experienced pathologists, who were blinded to the background information of the mice. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0%), 1 (1–10%), 2 (11–50%) and 3 (>50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A final immunoreactive score (IRS) was obtained for each case by multiplying the percentage and the intensity score.
8
Antibody Characterization for Immunoblotting and Immunocytochemistry
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The following antibodies were used for immunoblotting and immunocytochemistry: FLAG (mouse, Sigma F3165, 1:500; RRID:AB_259529), GFP (rabbit; Synaptic Systems 132 002, 1:1000; RRID:AB_887725), GAPDH (mouse; Millipore MAB374, 1:5000; RRID:AB_2107445), GM130 (mouse; BD Biosciences 610822, 1:1000; RRID:AB_398141), mCherry (rabbit; Abcam ab167453, 1:500; RRID:AB_2571870), Mint2 (rabbit; Sigma M3319, 1:1000; RRID:AB_477178), alpha 1 Sodium Potassium ATPase antibody (rabbit; Abcam ab7671, 1:200; RRID:AB_306023), Neurexin-1 (rabbit; Synaptic Systems 175 103, 1:1000; RRID:AB_10697816), SMI-312 (mouse; Abcam ab24574, 1:1000; RRID:AB_448151), Synapsin (rabbit; kind gift from Dr. Thomas S üdhof, P610, 1:500), Synaptobrevin (mouse; Synaptic Systems 104 211, 1:1000; RRID:AB_2619758), alpha-tubulin (mouse; Cell Signaling 3873, 1:1000; RRID:AB_1904178).
9
Confocal Imaging of Na+/K+-ATPase in Cardiomyocytes
10
Mitochondrial Protein Extraction and Analysis
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Mitochondrial proteins were extracted from homogenized hearts with a dounce potter in lysis buffer (sucrose 250 mM, K+ Hepes pH 7.5 20 mM, KCl 10 mM, MgCl2 1.5 mM, EDTA 0.1 mM, EGTA 1 mM, protease inhibitors, NaF 50 mM, Na3VO4 0.2 mM, PMSF 100 μM, DTT 1 mM, digitonin 0.025%) as previously reported [17 (link),53 (link),54 (link)]. The protein yield was quantified with the Bio-Rad protein assay (BIO-RAD, Milan, Italy). Western blot analysis for Cx43 or pCx43 was performed as described above. Primary antibody anti-TOM20 (Santa Cruz: SC-11415) was used as loading control. In order to verify the purity of mitochondrial protein extraction, a Western blot analysis was performed to evaluate the presence of proteins expressed only in the mitochondria (ox-Phos Complex II, Abcam, Cambridge, UK: ab14715) and the absence of proteins expressed in other cellular compartments (Na+/K+ ATPase, Abcam: ab7671) [55 (link)].
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