Western blot analysis was performed as described previously (12 (link)) using the following antibodies: mouse anti-AKT (BD Biosciences), rabbit anti-pAKT, rabbit anti-p4E-BP1, rabbit anti-4E-BP1, rabbit anti-pS6, mouse anti-S6, rabbit anti-pERK, rabbit anti-ERK, rabbit anti-pan-RAS (Cell Signaling, Beverly, MA, USA). Mouse anti-GAPDH (HyTest, Turku, Finland) or mouse anti-β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc.), and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL, USA) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Also, donkey anti-mouse IgG or donkey anti-rabbit (LI-COR Biotechnology, Bad Homburg, Germany) labeled with IRDye infrared dyes were used for detection. Representative blots of at least two independent experiments are shown.
Goat anti rabbit igg conjugated to horseradish peroxidase
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
Goat anti-rabbit IgG conjugated to horseradish peroxidase is a secondary antibody reagent used in various immunoassay and immunohistochemistry applications. It binds to rabbit primary antibodies and is conjugated to the enzyme horseradish peroxidase, which can be used to detect and visualize target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by Cytiva (11 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (11 mentions)
Mouse anti β actin, by Merck Group (6 mentions)
Mouse anti gapdh, by HyTest (5 mentions)
Mouse anti caspase 8, by Alexis Biochemicals (5 mentions)
Donkey anti goat igg, by Santa Cruz Biotechnology (4 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (4 mentions)
Goat anti mouse igg2b, by Southern Biotech (3 mentions)
Goat anti mouse igg1, by Southern Biotech (3 mentions)
Rabbit anti caspase 9, by Cell Signaling Technology (3 mentions)
Odyssey imaging system, by LI COR (3 mentions)
Mouse anti akt, by BD (2 mentions)
Rabbit anti p 4e bp1, by Cell Signaling Technology (2 mentions)
Rabbit anti 4e bp1, by Cell Signaling Technology (2 mentions)
Rabbit anti ps6, by Cell Signaling Technology (2 mentions)
Mouse anti s6, by Cell Signaling Technology (2 mentions)
Rabbit anti p erk, by Cell Signaling Technology (2 mentions)
Mouse anti caspase 9, by Cell Signaling Technology (2 mentions)
Mouse anti noxa, by Alexis Biochemicals (2 mentions)
Rat anti bmf, by Alexis Biochemicals (2 mentions)
16 protocols using goat anti rabbit igg conjugated to horseradish peroxidase
1
Western Blot Analysis of Protein Signaling
2
Western Blot Analysis of Apoptosis Regulators
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Western blot analysis was performed as described previously [52 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-NOXA, rat anti-BMF (Alexis Biochemicals, Grünberg, Germany), mouse anti-AKT, mouse anti-BCL-2, mouse anti-BAX, rabbit anti-BAK (BD Bioscience), rabbit anti-caspase-3, mouse anti-caspase-9, rabbit anti-pAKT, rabbit anti-p4E-BP1, rabbit anti-4E-BP1, rabbit anti-pS6, mouse anti-S6, (Cell Signaling, Beverly, MA), rabbit anti-MCL-1 (Stressgene Bioreagents, Victoria, BC). Mouse anti-GAPDH (HyTest, Turku, Finland) or mouse anti-β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA)) and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Representative blots of at least two independent experiments are shown.
3
BV6-Mediated Tumor Cell Killing
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BV6-mediated killing mechanism was analyzed in co-cultures of tumor and CIK cells. TE671 (TE) cells that were efficiently killed by combination therapy and grown in adherent cultures allowing a segregation of effector and target cells by several washing steps were used for analyses. Analyzed samples included TE and CIK cells alone, TE and CIK cells pre-incubated with 2.5 μMol BV6 for 4 h, TE cells co-cultured with CIK cells at an E:T ratio of 20:1 for 3 h, and TE cells pre-incubated with 2.5 μMol BV6 for 4 h followed by co-culture with CIK cells at an E:T ratio of 20:1 for 3 h. Co-cultures of TE and CIK cells were washed for the removal of CIK cells before TE cells were analyzed by western blot analysis. Western blot analysis was performed as described previously using the following antibodies: mouse anti-caspase-8 (1:1000; Alexis Biochemicals, Gruenberg, Germany), rabbit anti-Bid, rabbit anti-caspase-3, and rabbit anti-caspase-9 (1:1000; Cell Signaling, Beverly, MA, USA). Mouse anti-β-actin (1:10000; Sigma) was used as loading control (37 (link)). Goat anti-mouse IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany).
4
Western Blot Analysis of Apoptosis Regulators
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Western blot analysis was performed as described previously [28 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-Noxa, rat anti-Bmf (Alexis Biochemicals, Grünberg, Germany), mouse anti-Bcl-2, rabbit anti-Bcl-xL, mouse anti-Bax, rabbit anti-Bak (BD Transduction Laboratories, Heidelberg, Germany), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-Bim, mouse anti-PARP (Cell Signaling, Beverly, MA), acetylated histone H3 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-Mcl-1 (Stressgene, Victoria, BC), rabbit histone H3 (Abcam, Cambridge, UK). Mouse anti-GAPDH (HyTest, Turku, Finland), mouse α-Tubulin (Calbiochem, Darmstadt, Germany) or mouse β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) were used as secondary antibodies. Enhanced chemiluminescence (Amersham Bioscience, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odyssey Imaging System, LICOR Bioscience, Bad Homburg, Germany) were used for detection. Representative blots of at least two independent experiments are shown.
5
Investigating Apoptosis-Related Protein Expression
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Western blot analysis was performed as described previously40 (link) using the following antibodies: mouse anti-caspase-8, mouse anti-cFLIP (Alexis Biochemicals, Grünberg, Germany), mouse anti-FADD, mouse anti-XIAP (clone 28), mouse anti-RIP1 (BD Transduction Laboratories, Heidelberg, Germany), rabbit anti-Bid, rabbit anti-caspase-3, mouse anti-caspase-9 (Cell Signaling, Beverly, MA, USA), rabbit anti-TRAIL-R2 (Chemicon, Billerica, MA, USA), goat anti-cIAP1 (R&D Systems, Wiesbaden, Germany), rabbit anti-cIAP2 (Epitomics, Burlingname, CA, USA), mouse anti-TNFR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mouse anti-α-tubulin (Calbiochem, Darmstadt, Germany) or mouse anti-β-actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL, USA) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Representative blots of at least two independent experiments are shown.
6
Western Blot Analysis of Apoptotic Markers
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Western blot analysis was performed as described previously [28 (link)] using the following antibodies: BAK, BCL-2, BCL-XL (BD, New Jersey, USA), BCL-2 (Life Technologies, Inc.), caspase-3, caspase-9 (Cell Signaling, Beverly, MA), BAX NT, pH3 (Millipore, Darmstadt, Germany), MCL-1 (Enzo Life Science, Lörrach, Germany), GAPDH (HyTest, Turku, Finland). Goat anti-mouse IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) as secondary antibodies and enhanced chemiluminescence (Amersham Bioscience, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging were used for detection (Odyssey Imaging System, LI-COR Bioscience, Bad Homburg, Germany). Representative blots of at least two independent experiments are shown.
7
Western Blot Analysis of Signaling Proteins
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Western blot analysis was performed as previously described13 (link), and following antibodies were used:
Mouse anti-p-ERK 1/2 -p44/42 MAPK(Thr202/Tyr204) – Cell Signaling, Frankfurt, Germany
Rabbit anti-ERK 1/2 – Sigma-Aldrich
Rabbit anti-p-Akt (S473) – Cell Signaling
Mouse anti-Akt – BD Bioscience, Heidelberg, Germany
Mouse anti-GAPDH – Hytest Ltd., Turku, Finland
For chemiluminescent visualisation goat anti-mouse IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase at 1:5000 (Santa Cruz Biotechnology, Heidelberg, Germany) were used.
Mouse anti-p-ERK 1/2 -p44/42 MAPK(Thr202/Tyr204) – Cell Signaling, Frankfurt, Germany
Rabbit anti-ERK 1/2 – Sigma-Aldrich
Rabbit anti-p-Akt (S473) – Cell Signaling
Mouse anti-Akt – BD Bioscience, Heidelberg, Germany
Mouse anti-GAPDH – Hytest Ltd., Turku, Finland
For chemiluminescent visualisation goat anti-mouse IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase at 1:5000 (Santa Cruz Biotechnology, Heidelberg, Germany) were used.
8
Immunoblot Analysis of NF-κB Pathway
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Western blot analysis was performed as described previously using the following antibodies: mouse anti-phosphorylated IκBα (Cell Signaling, Danvers, MA, USA), rabbit anti-IκBα (Cell Signaling), rabbit anti-acetylated histone H3 (Millipore, Billerica, MA, USA), rabbit anti-NIK (Cell Signaling), mouse anti-p100/p52 (Millipore), rabbit anti-phosphorylated p65 (Cell Signaling) and rabbit anti-p65 (Abcam, Cambridge, MA, USA). Mouse anti-α-Tubulin (Sigma) and mouse anti-GAPDH (HyTest, Turku, Finland) were used as loading controls. Goat anti-mouse IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA) as secondary antibodies and enhanced chemiluminescence were used for detection (Amersham Bioscience, Freiburg, Germany). Alternatively, infrared dye-labeled secondary antibodies and infrared imaging were used for detection (Odyssey imaging system, LI-COR Bioscience, Bad Homburg, Germany). Representative blots of at least two independent experiments are shown.
9
Quantification of Antioxidant Enzymes
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Protein concentration of homogenate was determined using a colorimetric assay (BioRad Laboratories, Hercules, CA). The homogenate (20 µg) was electrophoresed using SDS-PAGE method and transferred to a nitrocellulose or PVDF membrane. The membrane was washed and then blocked in Blotto [5% milk, 10 mM Tris-HCl, 150 mM NaCl (pH 8.0), and 0.05% Tween 20] for 1 h at room temperature. An affinity purified rabbit anti-MnSOD antibody purchased from Upstate or anti-4-hydroxynonenal (4HNE) (Alexis) antibody was used. After two washings in TBST [10 mM Tris-HCl, 150 mM NaCl (pH 8.0), and 0.05% Tween 20], the blot was incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA) at a 1∶3000 dilution in Blotto for 1.5 h at room temperature. The blot was washed three times in TBST and once with TBS [10 mM Tris-HCl, and 150 mM NaCl (pH 8.0)]. Protein bands were visualized using the ECL detection system from Amersham Pharmacia Biotech (Buckinghamshire, England). An affinity purified sheep anti-CuZnSOD antibody (1∶3000), purchased from Calbiochem or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1∶3000) (Trevigen) was used for a loading control.
10
Western Blot Protein Analysis Protocol
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Briefly, whole protein samples were extracted, and their concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), following which 20 μg of protein was loaded and separated on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel. The membranes were incubated overnight at 4°C with specific primary antibodies MFGE8 (Abcam, dilution in 1:500) and GAPDH (Proteintech, dilution in 1:1000), followed by incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz, dilution in 1:5000) for 1 h at room temperature. The membranes were then visualized via a film following incubation with ECL solution (Cell Signaling Technology).
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