The following antibodies and reagents were used throughout the study: IP-10 (Peprotech 300–12), AMG487 (Tocris 448,710), mouse anti-human CXCR3-B specific antibody (Proteintech 60,065–1-Ig), mouse-anti human CXCR3 (R&D MAB160, recognizes both CXCR3-A and CXCR3-B), rabbit anti-human GAPDH (Cell Signaling 14C10), rabbit anti-human actin (Sigma A2668), mouse anti-human β-tubulin (Santa Cruz Biotech SC-101527), mouse-anti ddk tag (Origene TA50011–100), mouse anti-human E-cadherin (Invitrogen 135,700)-for immunofluorescence and rabbit anti-human E-cadherin (Cell Signaling 3195)-for immunoblotting and immunohistology.
Rabbit anti human gapdh
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-human GAPDH is a primary antibody that recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein from human samples. GAPDH is a widely expressed enzyme involved in glycolysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Amg487, by Bio-Techne (1 mentions)
Rabbit anti human e cadherin, by Cell Signaling Technology (1 mentions)
Ip 10, by Thermo Fisher Scientific (1 mentions)
Rabbit anti human actin, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Nanodrop 3000, by Thermo Fisher Scientific (1 mentions)
Rabbit anti hmga2, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg horseradish peroxidase hrp, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Donkey anti rabbit igg hrp, by GE Healthcare (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Sheep anti mouse igg hrp, by GE Healthcare (1 mentions)
14 protocols using rabbit anti human gapdh
1
Immunodetection of CXCR3 Isoforms
2
Quantification of Osteogenic Protein Expression
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Total protein extracted with Cell Lysis buffer (#9803; Cell Signaling Technology, Inc., Danvers, MA, USA) from osteogenic-induced hBMSCs was quantified using a Bicinchoninic Acid (BCA) kit (Thermo Fisher Scientific, Inc.). Following this, total protein was denatured by heating at 95°C for 5 min, mixed with loading buffer, and 30 µg proteins were separated by SDS-PAGE on 10% acrylamide gels. Proteins were subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% dried skimmed milk, and incubated with rabbit anti-human GAPDH (1:2,000; #2118; Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit anti-human Runx2 (1:1,000; ab48811; Abcam, Cambridge, MA, USA) antibodies at 4°C overnight and subsequently incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000; #7074; Cell Signaling Technology, Inc.) at 37°C for 1 h. Following chemiluminescence with a commercial assay (Pierce™ ECL Western Blotting substrate, #32106, Thermo Fisher Scientific, Inc.), protein bands were visualized by Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA). The bands were quantified using Quantity One software (version 4.52; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and normalized to GAPDH.
3
Western Blot Analysis of HMGA2 Protein
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Total proteins were isolated by RIPA buffer (Solarbio, Beijing, China) and quantified using a NanoDrop 3000 (Thermo Fisher Scientific). After subjected to Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein samples were transferred onto polyvinylidene fluoride membranes (Millipore, Bradford, MA, USA). Next, 5% fat-free milk buffer was used to block the membranes for 2 h at 37 °C. And then primary antibodies rabbit-anti-HMGA2 (1:500; Cell Signaling Technology, Danvers, MA, USA) or rabbit-anti-human GAPDH (1:1000; Cell Signaling Technology) was used to incubate the membranes overnight at 4 °C. The membranes were then washed with TBST and incubated with secondary antibody marked goat anti-rabbit IgG horseradish peroxidase (HRP) (1:2000; Cell Signaling Technology). After washing with TBST, the blots were detected using an ECL detection kit (Pierce Biotech, Rockford, IL, USA) and analyzed by Image Pro-Plus 6.0 software.
4
Immunoblotting Analysis of DNA Damage Signaling
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Whole cell extracts were prepared in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS and Roche complete mini protease inhibitors). Sample protein concentrations were determined by BCA assay (Pierce) and the same amount of protein was loaded into each well. Electrophoresed samples were transferred and immobilized onto PVDF membranes and immunoblotted according to antibody manufacturer instructions. The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A). The following secondary antibodies were used: goat anti-rabbit IgG-Alexa Fluor 488 (Life Technologies A11034), goat anti-mouse IgG-Alexa Fluor 488 (Life Technologies A11001), donkey anti-rabbit IgG-HRP (GE Healthcare NA934V) and sheep anti-mouse IgG-HRP (GE Healthcare NA931V).
5
Western Blot Analysis of ER Stress Markers
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Cells were lysed by RIPA Lysis buffer (Thermo Fisher Scientific), and total proteins were collected. After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), the membrane was blocked in 5% skim milk for 1 h and incubated with primary antibodies at 4°C overnight and secondary antibodies for 1 h. The primary antibodies used were rabbit antihuman CHOP (1 : 1000, sc793, Santa Cruz), rabbit antihuman eIF2α (1 : 1000, #9722, Cell Signaling Technology), rabbit antihuman p-eIF2α (1 : 1000, #9721, Cell Signaling Technology), and rabbit antihuman GAPDH (1 : 2000, #5174, Cell Signaling Technology). Bands were exposed to electrochemiluminescence (ECL).
6
Western Blot Analysis of CPT1A and PDH
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Cells were washed in cold PBS and resuspended in RIPA lysis buffer containing 0.1% Tween-20 and supplemented with phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and phosphatase inhibitor cocktail. Samples were homogenized using a microcentrifuge tube pestle, and lysates snap frozen and stored at -80° until use. Protein concentrations were quantified by bicinchoninic acid assay (Pierce Biotechnology).
Whole cell lysates were run on 4-12% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes, blocked using Intercept blocking buffer (Li-Cor), and probed with mouse anti-human CPT1A (Abcam), rabbit anti-human phospho-PDH (Cell Signaling Technology), or rabbit anti-human PDH antibodies (Cell Signaling Technology). Rabbit anti-human GAPDH and mouse anti-human alpha tubulin were included as loading controls for CPT1a and PDH/pPDH respectively (Cell Signaling Technology). Blots were then probed with anti-rabbit DyLight 800 and anti-mouse DyLight 488 secondary antibodies (Invitrogen) followed by multiplex fluorescent imaging using Bio-Rad Chemidoc Touch Imaging System (Bio -Rad, Hercules CA). Densitometric analysis of bands was performed with ImageJ. CPT1A expression was normalized to GAPDH. Phosphorylated PDH and PDH were normalized to alpha tubulin.
Whole cell lysates were run on 4-12% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes, blocked using Intercept blocking buffer (Li-Cor), and probed with mouse anti-human CPT1A (Abcam), rabbit anti-human phospho-PDH (Cell Signaling Technology), or rabbit anti-human PDH antibodies (Cell Signaling Technology). Rabbit anti-human GAPDH and mouse anti-human alpha tubulin were included as loading controls for CPT1a and PDH/pPDH respectively (Cell Signaling Technology). Blots were then probed with anti-rabbit DyLight 800 and anti-mouse DyLight 488 secondary antibodies (Invitrogen) followed by multiplex fluorescent imaging using Bio-Rad Chemidoc Touch Imaging System (Bio -Rad, Hercules CA). Densitometric analysis of bands was performed with ImageJ. CPT1A expression was normalized to GAPDH. Phosphorylated PDH and PDH were normalized to alpha tubulin.
7
Quantification of Placental Protein Profiles
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The protein concentration in cleared lysates from embryonic, placental, and newborn tissues was determined by BCA assay (Pierce, Rockford, IL). 80 µg of total protein was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human matriptase (R&D Systems), mouse anti-cow desmoglein 1 and 2 (Fitzgerald Industries International, Acton, MA), mouse anti-human Gcm1 (Abcam, Cambridge, MA), rabbit anti-human syncytinA (SantaCruz Biotechnology), rabbit anti-human pan-cadherin, rabbit anti-human GAPDH (both Cell Signaling Technology, Danvers, MA), rabbit anti-human claudin-1, and mouse anti-human claudin-2 (both Invitrogen, Carlsbad, CA) primary antibodies, and goat anti-mouse, mouse anti-rabbit (both DakoCytomation), or donkey anti-sheep (Sigma-Aldrich) secondary antibodies conjugated to alkaline phosphatase. Alkaline phosphatase activity was then visualized using nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate substrates. Where indicated, protein signal quantification was performed using ImageJ 1.46r software.
8
Immunoblotting for Autophagy Markers
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Mouse anti-human SQSTM1/p62 (D5L7G) (Cell Signaling, Danvers, 88588, MA, USA), rabbit anti-human GAPDH (Cell Signaling 2118, MA, USA) and rabbit anti-human LC3A/B (Cell Signaling 4108, MA, USA).
9
Western Blot Analysis of NLRP6 Protein
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The tissue was rinsed in saline and processed in RIPA or urea buffers. Protein samples were separated on SDS–PAGE gels under reducing conditions and transferred to nitrocellulose membranes, blocked for 1 h in 5% BSA or milk proteins diluted in PBS or TBS containing 0.5% or 0.1% Tween 20, respectively. Following blocking, membranes were incubated at 4°C overnight with the following antibodies: rabbit anti-mouse Nlrp6 (E-20; Santa Cruz), mouse anti-human NLRP6 (Clint-1; Adipogen), rabbit anti-human GAPDH (Cell Signaling), mouse anti-human tubulin (Sigma-Aldrich), mouse anti-human NLRP6 (R&D Systems), rabbit anti-GFP (Thermo Fisher Scientific), rabbit anti-mouse CD11b (Abcam), and mouse anti-mouse SMA (Clone 1A4; Sigma-Aldrich). Membranes were incubated for 1 h at room temperature with the secondary antibody at 1:5,000 in blocking buffer and visualized using ECL Western blotting detection reagents (Amersham, GE Healthcare). Images were captured using a ChemiDoc MP imaging device (Bio-Rad Laboratories).
10
Hirsutanol A Induces Apoptosis in Cells
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Hirsutanol A, a sesquiterpenoid compound isolated and synthesized in the laboratory of Professor Lan Wenjian, School of Pharmacy, Sun Yat-sen University, China, was dissolved in DMSO and stored in a refrigerator at -40˚C until use. p53 inhibitor pifithrin-α (PFTα) was purchased from APeXBIO Technology LLC. The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc. Hoechst 33258 was purchased from Beijing Solarbio Science & Technology Co., Ltd. An apoptosis test kit (Annexin V-FITC Apoptosis Detection Kit I) was purchased from BD Biosciences, and the cell cycle detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd. Rabbit anti-human p53 (1:1,000; cat. no. 2527), p53 upregulated modulator of apoptosis (Puma; 1:1,000; cat. no. 12450), Bcl-2 (1:1,000; cat. no. 4223), Caspase-3 (1:1,000; cat. no. 9662), poly (ADP-ribose) polymerase (PARP; 1:1,000; cat. no. 9532), cleaved caspase-3 (cCaspase-3; 1:1,000; cat. no. 9664) and cleaved poly(ADP-ribose) polymerase (cPARP; 1:1,000; cat. no. 9185) monoclonal antibodies were purchased from Cell Signaling Technology, Inc. The rabbit anti-human GAPDH (1:1,000; cat. no. 10494-1-AP) primary antibody was purchased from ProteinTech Group, Inc. The horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1,000; cat. no. A0208) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.
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