HUVECs were lysed with lysis buffer RIPA (G2002; Wuhan Goodbio Biotechnology Co., Ltd., Wuhan, China). Protein quantification was conducted using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The following primary antibodies were used: Cleaved-caspase-3 (9664), caspase-3 (9665), B-cell lymphoma 2-associated X protein (Bax; 2722), and Bcl-2 (2870) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), TLR4 (sc-10741; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p-TAK1 (4508), p-TBK1 (5483), p-p65 (3033), TAK1 (5206), TBK1 (3013), p65 (8242), and GAPDH (2118) (all from Cell Signaling Technology, Inc.). The corresponding peroxidase-conjugated secondary antibodies used were IRdye 800CW-conjugated goat anti-mouse immunoglobulin G (926–32210) and IRDye 800CW-conjugated goat anti-rabbit immunoglobulin G (926–32211) (both from LI-COR Biosciences, Lincoln, NE, USA). The blots were scanned using a two-color infrared imaging system (Odyssey; LI-COR Biosciences) and the expression of target proteins were normalized to the expression of GAPDH.
Irdye 800cw conjugated goat anti rabbit immunoglobulin g
Manufactured by LI COR
Sourced in United States
IRDye 800CW-conjugated goat anti-rabbit immunoglobulin G is a secondary antibody conjugated with the near-infrared fluorescent dye IRDye 800CW. It is designed for detection and quantification of rabbit primary antibodies in Western blotting, immunohistochemistry, and other fluorescence-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by LI COR (1 mentions)
Bcl 2 2870, by Cell Signaling Technology (1 mentions)
Gapdh 2118, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ku60019, by AstraZeneca (1 mentions)
Etoposide, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Neocarzinostatin, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Ve 821, by Selleck Chemicals (1 mentions)
3 protocols using irdye 800cw conjugated goat anti rabbit immunoglobulin g
1
Apoptosis Signaling Pathway Regulation
2
Assessing Contact Activation by HK Cleavage
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Cleavage of HK as a marker of contact activation27-29 (link) was assessed by immunoblot. Briefly, 0.25 μL of NHP, pretreated or not with cancer cell–derived EVs preincubated in the absence or presence of calf intestinal alkaline phosphatase (CIP) or corn trypsin inhibitor (CTI), was analyzed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. After transfer to polyvinylidene difluoride, membranes were blotted using affinity-purified rabbit antibodies raised against a peptide corresponding to a sequence in domain 5 of the human HK light chain (DHGHKHKHGHGHGKHKNKGKKN) that recognizes intact HK (∼120 kDa) and the free cHK light chain (54 kDa, 47 kDa) or antibodies raised to an epitope (CQPLGMISLMK) in the C terminus of the HK heavy chain exposed after cleavage of HK to cHK (62 kDa) (supplemental Figure 1). Bound antibodies were detected using a 1:15 000 dilution of IRDye 800CW–conjugated goat anti-rabbit immunoglobulin G (LI-COR Biosciences). The membrane was scanned using an Odyssey CLx imager, and relative amounts of intact HK and cHK were quantified using Image Studio Lite 5.2.
3
DNA Damage Response Pathway Inhibitors
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Inhibitors of DNA-PK ((DNA-dependent protein kinase; NU-7441; Calbiochem, currently Millipore, Billerica, MA, USA), ATM (KU60019; AstraZeneca, Wilmington, DE, USA) and ATR (VE-821; Selleck Chemicals, Houston, TX, USA) was stored in dimethyl sulfoxide at −80 °C. The DSB-inducing agents neocarzinostatin, etoposide and doxorubicin were all from Sigma-Aldrich (St Louis, MO, USA).
Source of antibodies are as follows: ATM phospho-Ser1981 and ATM (Millipore), SMC1 and SMC1 phospho-Ser966 (Abcam, Cambridge, MA, USA), KAP1 (KRAB-associated protein 1; BD Biosciences, San Jose, CA, USA), KAP1 phospho-Ser824 (Bethyl Laboratories, Montgomery, TX, USA) and β-actin (Sigma-Aldrich). IR DYE 680RD-conjugated goat anti-mouse immunoglobulin G and IR DYE 800CW-conjugated goat anti-rabbit immunoglobulin G (LI-COR, Lincoln, NE, USA).
Source of antibodies are as follows: ATM phospho-Ser1981 and ATM (Millipore), SMC1 and SMC1 phospho-Ser966 (Abcam, Cambridge, MA, USA), KAP1 (KRAB-associated protein 1; BD Biosciences, San Jose, CA, USA), KAP1 phospho-Ser824 (Bethyl Laboratories, Montgomery, TX, USA) and β-actin (Sigma-Aldrich). IR DYE 680RD-conjugated goat anti-mouse immunoglobulin G and IR DYE 800CW-conjugated goat anti-rabbit immunoglobulin G (LI-COR, Lincoln, NE, USA).
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