To analyze Rap1A expression in cells, we prepared cell lysates at 75% of confluence using 500 μL of radioimmunoprecipitation assay buffer (RIPA, 25 mmol/L Tris–HCl at pH 7.6, 150 mmol/L NaCl, 1% Nonidet P‐40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Protein concentrations of the lysates were determined with a Bio‐Rad protein assay kit (Hercules, CA). Immunoblotting analyses were performed as described previously. Antibodies against the following proteins were obtained from Santa Cruz Biotechnology: Rap1A, GAPDH. Antibodies against the following proteins were from Cell Signaling Technology (Danvers, MA): MEK1/2, ERK1/2, p38, pMEK1/2 (Ser217/221), pERK1/2 (Thr202/Tyr204), and pp38 (Thr180/Tyr182). 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, Buckinghamshire, UK). Immunoblotting reagents were from an electrochemiluminescence kit (Amersham Biosciences). Cells treated with U0126 (ERK inhibitor, Sigma) at indicated concentrations were also analyzed, using the above methods.
F ab 2 fragment of donkey anti mouse immunoglobulin
Manufactured by Cytiva
Sourced in United Kingdom
The F(ab)2 fragment of donkey anti-mouse immunoglobulin is a laboratory reagent designed for use in various immunoassay and immunodetection applications. It is a purified preparation of the F(ab)2 portion of donkey-derived antibodies specific to mouse immunoglobulins. This product can be used to detect, quantify, or capture mouse antibodies in samples.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase, by Cytiva (2 mentions)
Electrochemiluminescence kit, by Cytiva (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Donkey anti rabbit immunoglobulin, by Cytiva (2 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Rap1a, by Santa Cruz Biotechnology (1 mentions)
P mek1 2 ser217 221, by Cell Signaling Technology (1 mentions)
Mek1 2, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
U0126, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
P smad, by Cell Signaling Technology (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
P fak, by Cell Signaling Technology (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho erk1 2 4376, 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)
3 protocols using f ab 2 fragment of donkey anti mouse immunoglobulin
1
Western Blotting Analysis of Rap1A Expression
2
Immunoblot Analysis of Cell Signaling
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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).
3
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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