Cell lysates were prepared at 75% of confluence by using 500 μL of radio-immunoprecipitation assay buffer (25 mM Tris-HCl at pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) in 10-cm culture dishes with a 20-minute incubation on ice. The protein concentrations of the lysates were measured with a Bio-Rad protein assay kit (Hercules, CA). Immunoblot analyses were performed as previously described [37 (link), 38 (link)]. β-Actin antibodies were obtained from Santa Cruz Biotechnology. HER2, Claudin-1, ZEB1, ZO-1, Fak, pFak, Smad, pSmad were purchased from Cell Signaling Technology. The secondary antibodies were the F(ab)2 fragment of donkey anti-mouse immunoglobulin (product NA931) or of donkey anti-rabbit immunoglobulin (product NA9340) linked to horseradish peroxidase from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The immunoblot reagents were from an electrochemiluminescence kit (Amersham Biosciences, NJ, USA).
Donkey anti rabbit immunoglobulin
Manufactured by Cytiva
Sourced in United Kingdom
Donkey anti-rabbit immunoglobulin is a secondary antibody used in various immunoassays and detection methods. It recognizes and binds to rabbit primary antibodies, allowing for the visualization and detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
F ab 2 fragment of donkey anti mouse immunoglobulin, by Cytiva (2 mentions)
Horseradish peroxidase, by Cytiva (1 mentions)
Electrochemiluminescence kit, by Cytiva (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
P smad, by Cell Signaling Technology (1 mentions)
P fak, by Cell Signaling Technology (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
Phospho erk1 2 4376, by Cell Signaling Technology (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Mek1 2 9126, by Cell Signaling Technology (1 mentions)
Phospho mek1 2 2338, by Cell Signaling Technology (1 mentions)
Antibody against e cadherin, by BD (1 mentions)
2 protocols using donkey anti rabbit immunoglobulin
1
Immunoblot Analysis of Cell Signaling
2
Protein Expression Profiling for Cell Lines
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Total protein extract for each cell line was obtained by using a lysis buffer as described elsewhere (19 (link)), and equal amounts (30 µg/load) were analyzed by immuno-blotting. The antibody against β-actin was obtained from Sigma-Aldrich (A5441, 1:20,000). Antibodies against Bak (sc-832, 1:1,000), Bcl-2 (sc-7382, 1:500), matrix metalloproteinase (MMP)-9 (sc-6840, 1:1,000), MMP-2 (sc-13594, 1:1,000), vascular endothelial growth factor (VEGF, sc-507, 1:1,000) and JNK (sc-571, 1:1,000) were from Santa Cruz Biotechnology. Antibodies against ERK1/2 (9102, 1:1,000), phospho-ERK1/2 (4376, 1:1,000), MEK1/2 (9126, 1:1,000), phospho-MEK1/2 (2338, 1:1,000) and phospho-JNK (4668, 1:1,000) were obtained from Cell Signaling Technology. The antibody against Bcl-XL (#AM05, 1:1,000) was from Calbiochem. The antibody against CCL28 (18214-1-AP, 1:500) was from Proteintech. The antibody against E-cadherin (BD 610182, 1:1,000) was obtained from BD Labware (Franklin Lakes, NJ, USA). The secondary antibodies were F(ab)2 fragment of donkey anti-mouse immunoglobulin (product NA931) or of donkey anti-rabbit immunoglobulin (product NA9340) linked to horseradish peroxidase from Amersham Biosciences (Little Chalfont, Buckghamshire, UK).
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