Cells were lysed in phospho-lysis buffer (50 mM HEPES, pH 7.9; 150 mM NaCl; 4 mM Na Pyrophosphate; 1 mM EDTA, pH 8.0; 10 mM NaF; 0.5% Triton-X 100; 10% glycerol) with 1 mM PMSF, protease inhibitor cocktail set V (EMD Millipore) and phosphatase inhibitor cocktail set I (EMD Millipore) added freshly. Cell lysates were subject to SDS-PAGE and then transferred to an Immobilon-P membrane (Millipore). Membranes were then subjected to blocking in 5% nonfat dry milk in 1× TBST (20 mM Tris-HCL, pH 7.5; 150 mM NaCl; and 0.5% Tween 20) for 1 hour. Phospho-EIF4E Ser209 antibody was purchased from Cell Signaling. HSP90 and EIF4E antibodies were purchased from Santa Cruz Biotechnology. Primary antibodies were added to the membrane in 1× TBST over-night. The membrane was then washed 3 times in 1× TBST and HRP-linked anti-rabbit (GE Healthcare) or anti-mouse secondary antibody (Bio-Rad) was added for 1 hour. Blots were washed 3 times in 1× TBST and then developed using Amersham ECL western blotting detection reagent (GE Healthcare Life Sciences) per the manufacturer’s instructions. Chemiluminescence was detected using autoradiography film. Films were digitally scanned with Adobe Photoshop CS5 using a Canon CanoScan 8800F scanner.
Hrp linked anti rabbit
Manufactured by GE Healthcare
Sourced in United States
The HRP-linked anti-rabbit is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and visualize primary antibodies raised in rabbits. The HRP label enables colorimetric or chemiluminescent detection in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Canoscan 8800f scanner, by Canon (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail set 5, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail set 1, by Merck Group (1 mentions)
Phospho eif4e ser209 antibody, by Cell Signaling Technology (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Anti mouse secondary antibody, by Bio-Rad (1 mentions)
Hrp linked anti mouse, by GE Healthcare (1 mentions)
Amersham ecl, by GE Healthcare (1 mentions)
Na934, by GE Healthcare (1 mentions)
Anti pan akt, by New England Biolabs (1 mentions)
Anti phospho akt, by New England Biolabs (1 mentions)
C abl, by Cell Signaling Technology (1 mentions)
Raf 1, by Cell Signaling Technology (1 mentions)
Mouse monoclonal antibody against gapdh 6c5, by Abcam (1 mentions)
Sapk jnk, by Cell Signaling Technology (1 mentions)
Mark3, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated antibody, by Merck Group (1 mentions)
Anti mouse igg, by GE Healthcare (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
4 protocols using hrp linked anti rabbit
1
Western Blot Analysis of Phospho-EIF4E
2
BRCA1 Detection by Western Blot
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BRCA1 detection using western blotting was performed as following. Briefly, the primary antibodies against BRCA1 (1:50 dilution) and against GAPDH (1:20 000 dilution) were used. GAPDH was used as a loading control. Washes were carried out with Tris-buffered Saline Tween 20 (TBST) buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 30 min with 5 buffer changes. Secondary antibodies used were: Amersham ECL, HRP-linked anti-mouse (1:10 000 dilution) (GE Healthcare, #NA931-1ML) for BRCA1 and Amersham ECL, HRP-linked anti-rabbit (GE Healthcare, #NA934-1ML) (1:10 000 dilution) for GAPDH.
3
Protein Lysates and Wheat Germ Agglutinin Precipitation for DEP-1 Expression Analysis
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Preparation of protein lysates and wheat germ agglutinin precipitation (for DEP-1 expression analyses) were performed as described in Ref. [31] (link). Immunoblotting was done by standard protocols with primary antibodies: anti-phospho insulin receptor (IR) Y 972 (ab5678, 1:5000), anti-phospho IR Y 1158 (ab78355, 1:1000), anti-phospho IR Y 1361 (ab60946, 1:1000) (Abcam, Cambridge, UK), anti-DEP-1 (AF1934, 1:1000 of 1 μg/μl dilution, R&D Systems, Wiesbaden, Germany), anti-phospho Akt (#4060, 1:2000, Ser 473), anti-phospho Akt (#9275, 1:2000, Thr 308), anti-pan Akt (#9272, 1:1000) and anti-IR (#3025, 1:1000, 4B8) (Cell Signaling/New England Biolabs, Frankfurt, Germany). Secondary antibodies used were: HRP-linked anti-rabbit (NA934, 1:10,000, GE Healthcare), HRP-linked anti-goat (P 0160, 1:2000, Dako). Densitometric analyses were performed using ImageJ 1.46r.
4
Comprehensive Antibody Resource for Signaling
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A rabbit polyclonal antibody against the 19-kDa component of Oplophorus luciferase was kindly provided by Dr. Satoshi Inouye (Yokohama Research Center, JNC Corporation, Yokohama, Japan). Mouse monoclonal anti-FLAG peptide (clone M2) and horseradish peroxidase-conjugated antibodies were purchased from Sigma-Aldrich. Mouse monoclonal antibodies against SRPK1, SRPK2 (23/SRPK2), and CDK9 (D-7) were purchased from Pharmingen (BD Biosciences, San Jose, CA, USA), BD Transduction Laboratories (BD Biosciences), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Mouse monoclonal antibody against GAPDH (6C5) and rabbit polyclonal antibodies against CLK1 and Haspin were obtained from Abcam (Cambridge, UK). Rabbit monoclonal antibodies against CDC37 (D11A3), GSK3β (27C10), AKT1 (C73H10), MEK1 (30C8), SRC (32G6) and rabbit polyclonal antibodies against DYRK1A, DYRK1B, p42/p44 MAPK (ERK1/2), p38 MAPK, SAPK/JNK, p70S6K, CHK2, MARK3, CK1, CK2α, RAF1, FAK, FYN, c-ABL, and IKKα were purchased from Cell Signaling Technology (Beverly, MA, USA). These commercially available 1st antibodies and its reference numbers are listed in Table S2 . HRP-linked anti-rabbit and anti-mouse IgG were purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA) and Abcam, respectively.
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