Lung cryosections were stained with anti-CD3-Cy3 (17A2), anti-B220-AlexaFluor647 (RA3-GB2) (all prepared in-house) and anti-CD21/35-FITC (7G6; BD) antibodies. For chemokine staining, goat anti-CXCL12 (polyclonal, PeproTech) and goat anti-CXCL13 (R&D Systems) antibodies were detected with donkey anti-goat HRP (Jackson ImmunoResearch) and amplified with a Tyramide Signal Amplification system (PerkinElmer). Imaging was performed on an epifluorescence microscope equipped with an F-View II camera (Olympus). The numbers of BALT structures per section were counted using the polygon tool in cellSens software (Olympus).
Donkey anti goat hrp
Manufactured by Jackson ImmunoResearch
Sourced in United States
Donkey anti-goat HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and quantify goat primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit hrp, by Jackson ImmunoResearch (4 mentions)
Donkey anti mouse hrp, by Jackson ImmunoResearch (3 mentions)
Donkey anti rabbit hrp, by Jackson ImmunoResearch (2 mentions)
Sample loading buffer, by Thermo Fisher Scientific (2 mentions)
Sample reducing agent, by Thermo Fisher Scientific (2 mentions)
Streptavidin horseradish peroxidase hrp, by Jackson ImmunoResearch (2 mentions)
Acrylamide gradient, by Thermo Fisher Scientific (2 mentions)
Iblot2 apparatus, by Thermo Fisher Scientific (2 mentions)
Anti human igg biotinylatedantibody, by Southern Biotech (2 mentions)
Unlabeled goatanti human igm, by Southern Biotech (2 mentions)
Alas4000 imager, by GE Healthcare (2 mentions)
Donkey anti goat hrp, by R&D Systems (2 mentions)
Anti g6pdh, by Merck Group (2 mentions)
Image studio lite, by LI COR (2 mentions)
Luminata forte western hrp substrate, by Merck Group (2 mentions)
Genesnap, by Syngene (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tyramide signal amplification system, by PerkinElmer (1 mentions)
Goat anti cxcl13, by R&D Systems (1 mentions)
Cellsens software, by Olympus (1 mentions)
12 protocols using donkey anti goat hrp
1
Quantification of Bronchus-Associated Lymphoid Tissue
2
Quantifying Myc-Neurog2 Protein Levels
3
Antibody Isotype Detection by Western Blot
4
Antibody Isotype Detection by Western Blot
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B cell supernatants containing secreted antibodies were diluted in
H2O, 4x sample loading buffer (Life Technologies) and 10x sample reducing agent
(Life Technologies) and loaded onto precast gels with a 4-12% acrylamide gradient
(Invitrogen). The iBlot2 apparatus (Life Technologies) was used for protein transfer to
PVDF membranes followed by blocking for 1 h at room temperature with 3% BSA in TBS. The
membrane was incubated with different combinations of primary and secondary antibodies
diluted in TBS/1% BSA for 1 h at room temperature with 2 sequential TBS incubations to
wash the membrane between incubations. For detection of IgG, anti-human IgG-biotinylated
antibody (Southern Biotech, 2040-08) was used at 1 µg ml-1, followed by
25 ng ml-1 streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResearch,
016-030-084). IgM isotypes were stained with 10 µg ml-1 unlabeled goat
anti-human IgM (Southern Biotech, 2020-01) and 8 ng ml-1 donkey anti-goat HRP
(Jackson ImmunoResearch, 705-036-147). To detect LAIR1-containing antibodies, a polyclonal
goat anti-human LAIR1 antibody (R&D) at 2 µg ml-1 was combined
with secondary donkey anti-goat HRP. Membranes were developed with ECL-substrate on a
Las4000 imager (General Electric Company).
H2O, 4x sample loading buffer (Life Technologies) and 10x sample reducing agent
(Life Technologies) and loaded onto precast gels with a 4-12% acrylamide gradient
(Invitrogen). The iBlot2 apparatus (Life Technologies) was used for protein transfer to
PVDF membranes followed by blocking for 1 h at room temperature with 3% BSA in TBS. The
membrane was incubated with different combinations of primary and secondary antibodies
diluted in TBS/1% BSA for 1 h at room temperature with 2 sequential TBS incubations to
wash the membrane between incubations. For detection of IgG, anti-human IgG-biotinylated
antibody (Southern Biotech, 2040-08) was used at 1 µg ml-1, followed by
25 ng ml-1 streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResearch,
016-030-084). IgM isotypes were stained with 10 µg ml-1 unlabeled goat
anti-human IgM (Southern Biotech, 2020-01) and 8 ng ml-1 donkey anti-goat HRP
(Jackson ImmunoResearch, 705-036-147). To detect LAIR1-containing antibodies, a polyclonal
goat anti-human LAIR1 antibody (R&D) at 2 µg ml-1 was combined
with secondary donkey anti-goat HRP. Membranes were developed with ECL-substrate on a
Las4000 imager (General Electric Company).
5
Subcellular Fractionation and Western Blot Analysis
6
Immunofluorescence and Western Blotting Protocols
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Following primary antibodies were used for Immunofluorescence (IF) and Western blotting (WB): rabbit anti-GFP (1:200 IF, Invitrogen, A11122), chicken anti-GFP (1:200 IF, Aves, GFP-1020), rabbit anti-GFAP (1:100 IF, Dako, Z0334), mouse anti-NeuN (1:50 IF, Millipore, MAB377), rat anti-L1 (1:100 IF, Millipore, MAB5272), rabbit anti-Calretinin (1:500 IF, Millipore, MAB5054), goat anti-Nrp1 (2 µg/ml for function blocking, R and D, AF566), goat anti-VEGFR2 (1:15 IF, R and D, AF644), rabbit anti-VEGFR2 (1:1000 WB, Cell Signaling, 2479), rabbit anti-(phospho)VEGFR2 (Y1175) (1:500 WB, Cell Signaling, 2478), rabbit anti-(phospho/SFK (Y416) (1:200 IF, Invitrogen, 44660G), mouse anti-beta-III-tubulin (1:150 IF, Sigma, T5076), goat anti-VE-Cadherin (1:1000 WB, R and D, AF1002), Phalloidin (1:400 IF, Sigma, P1951). The following secondary antibodies were used: donkey anti-rabbit Alexa488 (Jackson Immunoresearch, 711-545-152), donkey anti-rabbit Alexa568 (Life Technologies, A10042), donkey anti-goat Alexa568 (Molecular Probes, A11057), goat anti-mouse Alexa568 (Invitrogen, A11031), goat anti-rat Alexa568 (Invitrogen, A11077), goat anti-chicken Alexa488 (Molecular Probes, A11039), donkey anti-goat HRP (Jackson Immunoresearch, 705–0350147), donkey anti-rabbit HRP (Jackson Immunoresearch, 711-035-152), donkey anti-mouse HRP (Jackson Immunoresearch, 715-035-150).
7
Immunocytochemistry and Western Blot Protocols
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For immunocytochemistry, the following antibodies were used: rabbit anti-Arc (custom-made, ProteinTech, Rosemont, IL, USA); DAPI (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-GluA1-NT (custom-made, generous gift from Dr. Richard Huganir; Widagdo et al., 2015 (link)); chicken anti-MAP2 (ab5392, Abcam, Cambridge, MA, USA); Alexa Fluor 488-, 555- and 647-conjugated secondary antibodies raised in donkey (Thermo Fisher Scientific; Jackson ImmunoResearch, West Grove, PA, USA). For Western blots, the following antibodies were used: rabbit anti-Arc (custom-made, ProteinTech); rabbit anti-E6AP (Ube3A; A300-352A, Bethyl Laboratories, Montgomery, TX, USA); goat anti-E6-AP (sc-8926, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-PSD-95 (75-028, clone K28/43, UC Davis/NIH NeuroMab Facility, Davis, CA, USA); goat anti-rabbit-HRP (111-035-003, Jackson ImmunoResearch); donkey anti-goat-HRP (705-035-003, Jackson ImmunoResearch); goat anti-mouse-HRP (115-035-003, Jackson ImmunoResearch). For immunoprecipitations, normal rabbit or mouse IgG (sc-2027 or sc-2025, Santa Cruz) was used as a control.
8
Immunocytochemistry and Western Blot Reagents
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Immunocytochemistry goat anti-ApoE (1:1000, Millipore, AB947), goat anti-Cathepsin B (1:1000, R&D systems, AF965), rabbit anti-P2X4 (1:200, Sigma, HPA039494), donkey anti-CD68 (1:300, Biorad, MCA1957A488T), mouse anti-6E10 (1:500, Biolegend, SIG-39320–0200), rabbit anti-Iba1 (Ionized calcium-binding adapter molecule 1, 1:2000, Wako, MNK4428), rat anti-P2X4 (1:200, kindly provided by Dr. Nolte [31 (link)]), donkey anti-rat-A594 (1:500, Jackson Immunoresearch, 712-586-150), donkey anti-goat-A488 (1:2000, Molecular probes, A11055), donkey anti-rabbit-A557 (1:2000, R&D systems, NL004), goat anti-rabbit-A488 (1:2000, Molecular probes, A11034).
Western blot mouse anti-tubulin (1:5000, Sigma, T9026), rabbit anti-HA (1:500, Invitrogen, 715,500), rabbit anti-P2X4 (1:500, Alomone Labs, APR-002), anti-HA beads (1:500 Santa-Cruz Biotechnology), horse anti-mouse-HRP (1:2000, Cell signaling, 7076S), goat anti-rabbit-HRP (1:2000, Jackson Immunoresearch, 111-035-144), donkey anti-goat-HRP (1:2000, Jackson Immunoresearch, 705–035-003).
Western blot mouse anti-tubulin (1:5000, Sigma, T9026), rabbit anti-HA (1:500, Invitrogen, 715,500), rabbit anti-P2X4 (1:500, Alomone Labs, APR-002), anti-HA beads (1:500 Santa-Cruz Biotechnology), horse anti-mouse-HRP (1:2000, Cell signaling, 7076S), goat anti-rabbit-HRP (1:2000, Jackson Immunoresearch, 111-035-144), donkey anti-goat-HRP (1:2000, Jackson Immunoresearch, 705–035-003).
9
Yeast Protein Extraction and Western Blot
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One ml of the yeast cell culture (1×107 cells) was treated with 10 M trichloroacetic acid (TCA) to a final concentration of 20% (v/v) for 10 min and centrifuged at full speed for 1 min. The resulting pellets were dissolved in 100 μl of 0.5% SDS, 42 mM Tris-HCl at pH 6.8. Then 300 μl of glass beads (Sartorius, BBI-8541701) were added, bead-beaten twice at maximum force for 30 sec and boiled for 5 min. Around 40 μg to 60 μg of protein from each sample was separated at 90 V (10% polyacrylamide/SDS gel) and transferred to PVDF membranes (Immobilon-P; Millipore). The primary antibodies used were anti-HA 1:100 (12CA5), anti-PAP 1:4.000 (Sigma, P1291), anti-PSTAIRE 1:5000 (Abcam, ab10345) and anti-G6PDH 1:500 (Sigma, A9521). The secondary antibodies used were donkey anti-goat-HRP, donkey anti-mouse-HRP and goat anti-rabbit-HRP 1:25000 (all from Jackson Laboratories). Immunoblots were developed using Luminata Forte Western HRP Substrate (Millipore) and images were taken using GeneSnap (Syngene) and quantified using Image Studio Lite (Li-Cor).
10
Immunoblotting of cellular proteins
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The primary antibodies used were anti tubulin 1:200 (Sigma T5168), anti FLAG 1:500 (Sigma F3165), anti Rad53 1:700 (Santa Cruz, sc-6749), and anti-G6PDH 1:500 (Sigma, A9521). The secondary antibodies used were sheep anti-mouse HRP 1:30.000, donkey anti-goat-HRP 1:20.000, and goat anti-rabbit-HRP 1:25.000 (all from Jackson laboratories). Immunoblots were developed using Luminata Forte Western HRP Substrate (Millipore) and images were taken with GeneSnap (Syngene) and quantified with Image Studio Lite (Li-Cor).
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