The rabbit phospho-site–specific GPR35 antiserum pSer300/pSer303-hGPR35a (Cat number (7TM0102C), raised against the sequence KAHKpSQDpSLCVTL, and the pSer298/pSer301-mGPR35 antiserum (7TM0102B), raised against the sequence TPHKpSQDpSQILSLT, were developed in collaboration with 7TM Antibodies GmbH. IRDye 800CW donkey anti-rabbit IgG, IRDye 800CW donkey anti-goat IgG, and IRDye 800CW goat anti-rat IgG were from LI-COR Biosciences. Alexa Fluor 488-goat anti-rabbit IgG and Alexa Fluor 594-donkey anti-rat IgG were from Abcam. Mouse monoclonal anti-FLAG M2 was from Merck. Horseradish peroxidase anti-mouse (sheep) was from GE Healthcare. High affinity anti-HA (rat) and anti-HA affinity matrix were from Roche Diagnostics.
Irdye 800cw donkey anti goat igg
Manufactured by LI COR
Sourced in United States
The IRDye 800CW donkey anti-goat IgG is a near-infrared fluorescent dye-labeled secondary antibody that binds to goat immunoglobulin G (IgG) for use in immunoassays and imaging applications. It provides a sensitive detection method for target proteins that have been labeled with a primary goat antibody.
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Lab products found in correlation
Irdye 800cw goat anti rat igg, by LI COR (4 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (4 mentions)
Irdye 800cw donkey anti rabbit igg, by LI COR (4 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (4 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Irdye 680lt donkey anti rabbit igg, by LI COR (3 mentions)
High affinity anti ha rat and anti ha affinity matrix, by Roche (2 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Starbright blue 700 goat anti rabbit igg, by Bio-Rad (2 mentions)
Fam83g, by Abcam (2 mentions)
Starbright blue 700 goat anti mouse igg, by Bio-Rad (2 mentions)
Lamin a c, by Cell Signaling Technology (2 mentions)
α tubulin, by Thermo Fisher Scientific (2 mentions)
Odyssey clx infrared imaging system, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sc 25778, by Santa Cruz Biotechnology (2 mentions)
Irdye 800cw donkey anti mouse igg, by LI COR (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
Ab81288, by Abcam (1 mentions)
25 protocols using irdye 800cw donkey anti goat igg
1
Phospho-specific GPR35 Antibody Characterization
2
Quantitative Western Blot Analysis of BDNF and TrkB
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HIP and PFC samples (20 µg total protein) were separated on 10% criterion TGX gels (Bio-Rad) and transferred to nitrocellulose membranes. The samples were then blocked in odyssey blocking buffer (LI-COR, Lincoln, NE, USA) and probed with the primary antibodies: rabbit anti-BDNF (Sigma AV41970; 1:2000), mouse anti-β-actin (926-42212, LI-COR; 1:3000), goat anti-TrkB (AF1494, R&D Systems, Minneapolis, MN, USA; 1:500), and rabbit anti-TrkB(Y817) (ab81288, Abcam, Cambridge, UK; 1:1000) overnight at 4 °C. This was followed by incubation with the appropriate IRDye conjugated secondary antibody for 1 h at RT: IRDye 800CW donkey anti-goat IgG, IRDye 680RD donkey anti-rabbit IgG, IRDye 800CW goat anti-rabbit IgG, or IRDye 680RD goat anti-mouse IgG, all in 1:15,000 dilution (LI-COR). Infrared signals were detected using the Odyssey CLx infrared imaging system (LI-COR, and bands were quantified using Image Studio software (LI-COR).
3
Antibody Identification for Signaling Pathway
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Anti-MLK3 (A-20) (for detection of murine MLK3), anti-FRA-1 (R-20), anti-JNK1/3 (C-17), anti-ERK1 (K-23), anti-P38 (C-20), anti-actin (C-2), anti-p-c-JUN (S63)(KM-1) and anti-c-JUN (H-79) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-MLK3 (C-terminal) (for detecting human MLK3) was from Epitomics (Cambridge, MA, USA). Anti-p-MLK3, anti-p-ERK-1/2 (T202/Y204)(E-10), anti-p-JNK1/2 (T183/Y185)(81E11) and anti-p-P38 (T180/Y182) (#9216) were obtained from Cell Signaling (Danvers, MA, USA). Anti-MMP-1 (#36665 R) was purchased from R&D systems (Minneapolis, MN, USA), anti-p-paxillin S178 (#A300-100 A) was purchased from Bethyl Laboratory (Montgomery, TX, USA). IRDye 800CW goat anti-mouse IgG, IRDye 680 goat anti-rabbit IgG and IRDye 800CW donkey anti-goat IgG were from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit IgG conjugated with Alexa Fluor 488 and 546 was from Invitrogen and used for immunofluorescence staining.
4
Analyzing HIV-1 Viral Particle Production
5
GFP Expression and Quantification in HeLa Cells
6
Phospho-specific GPR35 Antibody Characterization
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A rabbit phospho-site–specific hGPR35 antiserum pSer300/pSer303-hGPR35a (catalogue number 7TM0102C), raised against the sequence KAHKpSQDpSLCVTL, and a pSer298/pSer301-mGPR35 antiserum (7TM0102B), raised against the sequence TPHKpSQDpSQILSLT have been described previously (5 ). A GPR35 (nonphospho) antibody (cat number 7TM0102N), directed against the distal part of the carboxyl-terminal tail of hGPR35 was developed in collaboration with 7TM Antibodies GmbH. IRDye 800CW donkey anti-rabbit immunoglobulin G (IgG), IRDye 800CW donkey anti-goat IgG, and IRDye 800CW goat anti-rat IgG were from LI-COR Biosciences. Horseradish peroxidase anti-mouse (sheep) was from GE Healthcare. High-affinity anti-HA (rat) and anti-HA affinity matrix were from Roche Diagnostics. Anti-LgBiT mAb (cat number N7100) which is an affinity-purified mouse mAb for detection of LgBiT and LgBiT-fusion proteins by Western blotting was purchased from Promega Corporation. GRK isoform–directed antisera for GRK 2 sc-13143 (C-9) c and GRK 5 sc-518005 (D-9) were from Santa Cruz Biotechnology while GRK 3 CS #80362 (D8G6V) and GRK 6 CS #5878 (D1A4) were from Cell Signaling Technology. Further details are found in Reichel et al. (23 (link)).
7
RECQL Protein Detection Assay
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Cells were harvested and lysed in ELB buffer (150 mM NaCl; 50 mM Hepes, pH 7.5; 5 mM EDTA; 0.1% NP-40) containing protease inhibitors (Complete; Roche) for 30 min. Protein concentrations were determined using the BCA protein assay kit (Pierce). Primary antibody used to detect mouse RECQL was rabbit polyclonal anti-Recql (A300-450A; 1/1,000; Bethyl Laboratories). For detection of human RECQL, mouse monoclonal anti-Recql (A-9, Sc166388; 1/250; Santa Cruz) was used. For detection of actin, polyclonal goat anti-actin (I-19; 1/1,000; Santa Cruz) was used. Secondary antibodies used were IR Dye 800CW donkey antigoat IgG (LI-COR) and IR Dye 800CW goat-antimouse IgG (LI-COR).
8
Protein Quantification and Fractionation
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Protein lysates were prepared by homogenizing whole-brain tissue in cold lysis buffer containing phosphate-buffered saline, 1% Triton X-100, and protease inhibitors (Roche Complete, Mini), centrifugation at 5000 rpm for 10 min, followed by supernatant collection, and Western blotted as previously described (11 (link)). Synaptosomes and membrane fractions were prepared from whole brain as previously described (58 ). NAc punches (1.2 mm in diameter) around the anterior commissure were collected from coronal sections (thickness, 2 mm). Primary antibodies included rabbit anti-ASIC1 polyclonal antiserum (MTY19) diluted 1:3000 (59 (link)), rabbit anti-glutamate receptor 1 polyclonal antibody (Millipore, AB1504) diluted 1:1000, goat anti-CA4 polyclonal antibody (R&D Systems, AF2414) diluted 1:1000, mouse anti-PSD95 monoclonal antibody (clone K28/43, catalog no. 05-494, Upstate) diluted 1:300, rabbit anti-MBP monoclonal antibody (Abcam, 218001) diluted 1:1000, and mouse anti-GFAP (Millipore, MAB360) diluted 1:1000. Secondary antibodies (diluted 1:10,000 to 1:20,000) included IRDye 680LT donkey anti-rabbit immunoglobulin G (IgG; LI-COR, 926-68023), IRDye 800CW donkey anti-rabbit IgG (LI-COR, 926-32213), IRDye 800CW donkey anti-goat IgG (LI-COR, 926-32214), and IRDye 680LT donkey anti-mouse IgG (LI-COR, 926-68022). Membranes were imaged with an Odyssey imaging system (LI-COR).
9
IL-1β Secretion Assay in LPS-Stimulated IMG Cells
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IMG cells in a six-well poly-d -lysine-coated tissue culture dish were treated for 6 h with or without LPS (10 ng/mL) in 1 mL of full growth media at 37 °C 5 % CO2. Ac-YVAD-CMK (40 μM) was then added to the appropriate wells for 5 min prior to the addition of ATP (5 mM) for 30 min. The IMG cell-conditioned media were then collected and concentrated ten times from 1 mL to 100 μL using a 10K MWCO Nanosep Omega centrifugal device (PALL, Ann Arbor, MI). Seven microliters of this media was heated at 95 °C for 5 min and then resolved on a 4–20 % SDS-PAGE gel. The protein was then transferred onto a 0.2-μm nitrocellulose membrane using a wet-transfer apparatus at 100 V for 60 min. The membrane was blocked with 5 % milk in TBST at 4 °C for 1 h prior to incubation overnight at 4 °C with goat polyclonal IL-1β antibody (1:500 dilution; Santa Cruz Biotechnology, Inc.). The membrane was washed three times with TBST and then was incubated for 1 h at room temperature with IRDye 800CW donkey anti-goat IgG (1:5000 dilution; Li-Cor) in TBST 3 % milk. The membrane was washed three times with TBST and was imaged using Li-Cor Odyssey 2.1 infrared detection technology.
10
Western Blot Analysis of Transcription Factors
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Proteins were transferred to polyvinylidene difluoride membranes (MilliporeSigma, Cat #IPFL00010) and blocked using protein‐free blocking buffer (Thermo Fisher Scientific, Cat #23227). Membranes were probed with various anti‐TF antibodies shown in Table 1 . All primary antibodies were used at a 1:1000 dilution in blocking buffer. After washing with Tris‐buffered saline containing 0.1% Tween 20 (TBS‐T), membranes were incubated with either Alexa Fluor 647 chicken anti‐mouse IgG (Invitrogen, Cat #A21463), Alexa Fluor 680 rabbit anti‐goat IgG (Invitrogen, Cat #A21088), or IR Dye 680 RP goat anti‐rabbit IgG (LI‐COR Biosciences, Lincoln, NE, USA; Cat #926‐68071). All secondary antibodies were used at a 1:10 000 dilution in blocking buffer. Specific antigen‐antibody complexes were visualized using an Odyssey imaging system (LI‐COR Biosciences). Antibodies were removed with stripping buffer (Thermo Fisher Scientific, Cat #21059), and membranes were reprobed with either anti‐human GAPDH or anti‐mouse GAPDH antibodies. Both antibodies were diluted 1:1000 in blocking buffer. After washing with TBS‐T, membranes were incubated with either IR Dye 800 CW goat anti‐mouse IgG (LI‐COR Biosciences, Cat #926‐33210) or IR Dye 800 CW donkey anti‐goat IgG (LI‐COR Biosciences, Cat #926‐32214). An Odyssey imaging system was used to detect antibody‐protein complexes.
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