All immunoblotting and immunohistochemical staining procedures comply with recommendations detailed in the BJP editorial (Alexander et al., 2018 (link)). Protein samples (20 μg) were separated by SDS-PAGE on 10 % gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked by 5% non-fat dry milk (NFDM) or bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight at 4 °C with a primary antibody against TRPV1 (sc-12498, Santa Cruz, goat polyclonal IgG); 1:500 dilution in TBST with 5 % NFDM or BSA. Blots were then washed with TBST prior to the addition of an HRP-conjugated rabbit anti-goat IgG secondary antibody (1:10,000 dilution in TBST with 5 % NFDM or BSA) for 1 hour at room temperature. Membranes were developed using the ECL Prime reagent (Amersham) and scanned using an EPSON scanner.
Goat polyclonal igg
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom
Goat polyclonal IgG is a laboratory reagent used for various immunological and biochemical applications. It is a mixture of antibodies derived from goats that recognize multiple epitopes on target antigens. This product can be used as a capture or detection antibody in techniques such as ELISA, Western blotting, and immunoprecipitation.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting detection reagent, by Cytiva (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Sc 12498, by Santa Cruz Biotechnology (1 mentions)
Ecl prime reagent, by Cytiva (1 mentions)
Semi dry transfer apparatus, by Cytiva (1 mentions)
Tnt quick coupled transcription translation system, by Promega (1 mentions)
Wet blot transfer apparatus, by Bio-Rad (1 mentions)
Carbonate bicarbonate buffer, by Merck Group (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Fitc donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Anti brca1 igg, by Santa Cruz Biotechnology (1 mentions)
Rabbit polyclonal igg, by Santa Cruz Biotechnology (1 mentions)
Human il 6 elisa kit, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Odyssey blocking buffer pbs, by LI COR (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Ab18723, by Abcam (1 mentions)
Normal horse serum, by Jackson ImmunoResearch (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Vector Laboratories (1 mentions)
9 protocols using goat polyclonal igg
1
TRPV1 Protein Expression Analysis by Immunoblotting
2
Colon Protein Expression Analysis
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Total proteins isolated from colon were subjected to Western blot analysis. The concentration of total proteins was determined by BCA protein assay (Pierce, Rockford, IL). 40 micrograms of total proteins were separated in a 7.5–15% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane with a semidry transfer apparatus (Amersham Pharmacia, Piscataway, NJ). The membrane was blocked in 5% non-fat dry milk for 1 hour and probed overnight with a primary antibody. Subsequently the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The proteins were detected using ECL Western blotting detection reagents (Amersham-Pharmacia). Actin (43-kD) was used as loading control and identified using a goat polyclonal IgG (Santa Cruz Biotechnology Inc., Dallas, Texas). The following antibodies were used: Dclk1 (ab31704) (Abcam, Cambridge, MA), Claudin-1 (sc-137121), Claudin-5 (sc-28670), Claudin-7 (sc-17670) (Santa Cruz Biotechnology, Dallas, TX), E-Cadherin (MA5-15711) (Life Technologies, Carlsbad, CA), β-actin (4970), NF-κB p65 (D14E12) (Cell Signaling, Danvers, MA).
3
Non-Radioactive 21OH Variant Expression
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Non-radioactive TNT Quick Coupled Transcription/Translation system (Promega) with Sp6 RNA polymerase was used for in vitro protein expression. Expression of all the 21OH variants was verified by western blot using a primary antibody against 21OH (c17, goat polyclonal IgG, Santa Cruz Biotechnology) and alkaline phosphatase-conjugated donkey anti-goat IgG secondary antibody (Santa Cruz Biotechnology).
4
Immunoprecipitation and Western Blotting Protocol
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For immunoprecipitation studies, IEC extracts were normalized for protein concentration and pre-cleared with 30 μl of protein A/G-coated Sepharose beads for 1 h at 4 °C. Immunoprecipitation was carried out by incubating the fractions overnight at 4 °C with antibody recognizing Dclk1. Immune complexes were captured by incubation with 50 μl of protein A/G-Sepharose beads for 2 h at 4 °C. Control experiments were performed by conducting immunoprecipitations in the presence of the control IgG antisera. The immunoprecipitated proteins were recovered by boiling the Sepharose beads in 2X SDS sample buffer.
Total IEC extracts or immunoprecipitated proteins were subjected to SDS-PAGE. Twenty-five micrograms of the total protein were size-separated in a 7.5–15% SDS polyacrylamide gel and transferred electrophoretically onto a PVDF membrane with a wet blot transfer apparatus (Bio-Rad, Hercules, CA). The membrane was blocked in 5% non-fat dry milk for 1 h and probed overnight with a primary antibody. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The proteins were detected using ECL western blotting detection reagents (Amersham-Pharmacia, Piscataway, NJ). Actin (42-kD) was used as loading control and identified using a goat polyclonal IgG (Santa Cruz Biotechnology Inc., Dallas, Texas).
Total IEC extracts or immunoprecipitated proteins were subjected to SDS-PAGE. Twenty-five micrograms of the total protein were size-separated in a 7.5–15% SDS polyacrylamide gel and transferred electrophoretically onto a PVDF membrane with a wet blot transfer apparatus (Bio-Rad, Hercules, CA). The membrane was blocked in 5% non-fat dry milk for 1 h and probed overnight with a primary antibody. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The proteins were detected using ECL western blotting detection reagents (Amersham-Pharmacia, Piscataway, NJ). Actin (42-kD) was used as loading control and identified using a goat polyclonal IgG (Santa Cruz Biotechnology Inc., Dallas, Texas).
5
Antibody Depletion for Bactericidal Activity
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In a subset of samples representing all study arms and both TD and nTD groups, antibodies specific to H or O9:LPS, or total IgA or IgG antibodies were depleted from the serum to assess the contributions of each of these antibodies to the bactericidal activity in sera. Antibodies specific to H or O9:LPS were adsorbed on to 96-well Maxisorp plates (Nunc, Copenhagen, Denmark) pre-coated with 1 µg/mL H or 15 µg/mL O9:LPS antigen in carbonate bicarbonate buffer (Sigma, UK), for eight rounds of 30-min incubations. A plate coated with BSA was used to create mock depleted controls. Total IgA and IgG antibodies were depleted using the Dynabeads Antibody coupling kit (Life Technologies Limited, UK). Briefly, each diluted serum sample was incubated for 1 h with 2-mg magnetic beads coated with either 10 µg of goat polyclonal IgG antibody raised against human IgA or IgG (AbD Serotec, UK), or an goat polyclonal IgG (Santa Cruz Biotechnology, USA). The depletions resulted in approximately 56% (H), 87% (O9:LPS), 72% (IgA), and 93% (IgG) median reduction in the antibody titer, as measured with ELISA.
6
Quantitative Immunofluorescence Analysis of DNA Damage and Repair Proteins
7
Western Blot and ELISA for Detecting Protein Levels
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For protein detection by western blot, 50 μg of protein were separated in a polyacrylamide Mini-PROTEAN TGX Gel (Bio-Rad, Hercules, CA) and transferred to a PVDF membrane by semi-dry transfer using the iBlot Dry Blotting System (Thermo Fisher Scientific). The membrane was blocked for 1 h at 37°C with PBS Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE). Then, primary antibody was added and the membrane was incubated overnight at 4°C: Anti-p53 mouse monoclonal IgG2a (Santa Cruz Sc-126, 1:200 dilution) or Anti-actin (I-19) Goat polyclonal IgG (Santa Cruz Sc-1616, 1:4,000 dilution). After washing with 1x PBS (phosphate-buffered saline) Tween, the membrane was incubated for 1 h at 37°C with a fluorophore labeled secondary antibody: Anti-mouse donkey polyclonal IgG 800CW (926-32212, Li-COR, 1:15,000 dilution) or Anti-goat donkey polyclonal IgG 680RD (926-68074, Li-COR, 1:15,000 dilution). Finally, after washing, the membrane was scanned with the Odyssey Imaging System (Li-COR). ELISA experiments were performed following the manufacturer's instructions. IL-6 protein was measured in HaCaT, HaCaT-pLVX, HaCaT-16E6, HaCaT-18E6, HaCaT-62E6, HaCaT-84E6, HeLa, and SiHa cells supernatants using the Human IL-6 ELISA Kit (Thermo Fisher Scientific).
8
Immunohistochemical Detection of DCX
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Sections were brought to room temperature in tris buffered saline (TBS), followed by a 10-min wash in fresh TBS. Sections were then incubated for 30-min in a H₂O₂ solution (97% TBS, 1% methanol, 2% of 3% retail H₂O₂) to eliminate endogenous peroxidases. After three 5-min TBS rinses, non-specific binding was blocked with 5% normal horse serum (Jackson ImmunoResearch Laboratories Inc.) and 0.5% Triton X-100 (Sigma-Aldrich) in TBS for 30-min at room temperature. This was followed by exposure to anti-DCX antibody (goat polyclonal IgG, Santa Cruz Biotechnology; sc-8066, 1:150; or rabbit polyclonal IgG, Abcam; ab18723, 1:1000) in the same blocking buffer overnight at 4˚C. After three 5-min TBS rinses, sections were incubated for 3 h in biotinylated horse anti-goat (1:200, Vector Labs) or biotinylated goat anti-rabbit secondary antibody (1:200, Vector Labs) in TBS, rinsed again, and exposed for 1 h to an avidin-biotin complex (Vector Labs). Sections were rinsed in TBS and then reacted in a solution of 0.04% of 3,3’-diaminobenzidine tetrahydrochloride (DAB, Vector Labs) with nickel until the tissue changed color (~3–10 min). Following three 5-min TBS rinses, sections were dehydrated in ethanols, delipidized in xylenes, and cover slipped with Krystalon (Millipore-Sigma).
9
Isolation and Characterization of Progenitor Cells
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Freshly isolated BMMC were washed in phosphate-buffered saline (PBS) supplemented with 1% FCS and then incubated with anti-CD34, a marker of progenitor cells (1/25, goat polyclonal IgG, Santa Cruz Biotechnology, Inc.), anti-CD90, a marker of multipotent cells and fibroblasts (1/25, mouse monoclonal IgG, clone OX-7, Chemicon Int), anti-CD45, a pan-hematopoietic marker
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