Western blotting was performed as described previously43 (link). Membranes were blotted with rabbit monoclonal antibody against EGFP antigen (ab184601, 1:2,000, Abcam, Cambridge, UK) or with polyclonal antibody against α-tubulin (PM054–7, 1:5,000; MBL, Aichi, Japan) and the appropriate standard peroxidase-labeled anti-mouse IgG or anti-rabbit IgG secondary antibody, according to the manufacturer’s instructions (GE Healthcare). Immunoreactive bands were visualized using the ECL detection system (Pierce, Rockford, IL, USA). EGFP levels were quantified using ImageJ software.
Anti rabbit igg secondary antibody
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Japan
The Anti-rabbit IgG secondary antibody is a laboratory reagent used in various immunoassay techniques. It binds to rabbit primary antibodies, allowing for the detection and visualization of target proteins or molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated anti mouse igg, by GE Healthcare (3 mentions)
Vinculin, by Cell Signaling Technology (2 mentions)
Ptk2 fak, by Cell Signaling Technology (2 mentions)
Ha tag, by Cell Signaling Technology (2 mentions)
V5 tag, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Enhanced chemiluminescence substrate, by PerkinElmer (2 mentions)
Lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Bolt bis tris precast denaturing gels, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Horseradish peroxidase conjugated anti mouse igg, by GE Healthcare (2 mentions)
Novex ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (2 mentions)
Ab184601, by Abcam (1 mentions)
Peroxidase labeled anti mouse igg, by GE Healthcare (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Pvdf blocking reagent, by Toyobo (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Can get signal, by Toyobo (1 mentions)
14 protocols using anti rabbit igg secondary antibody
1
Western Blotting for EGFP Quantification
2
Immunoblotting of Protein Signaling
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Cells were washed with PBS twice, and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). Total protein lysates were run in Nupage 4% to 12% Bis-Tris gradient precast gels (Invitrogen) in MOPS buffer. Membranes were probed using specific antibodies: PTK2/FAK (#3285S; RRID: AB_2269034) phosphorylated AKT (pAKT) (S473; #4060; RRID: AB_2315049), pAKT (T308; #13038; RRID: AB_2629447), total AKT (#4691; RRID: AB_915783), pS6 (S240/244; #5364; RRID: AB_10694233), pS6 (S235/236; #4858; RRID: AB_916156), HA-tag (#3724; RRID: AB_1549585), V5-tag (#13202; RRID: AB_2687461), β-actin (#4970S; RRID: AB_2223172), and vinculin (#13901; RRID: AB_2728768) were purchased from Cell Signaling Technology. The same antibody was used to detect both PTK2/FAK (125 kDa) and FRNK (43 kDa). All primary antibodies were diluted 1:1,000 and anti-rabbit IgG secondary antibody (GE Healthcare; RRID: AB_772206) (1:10,000) was used.
3
Western Blot Analysis of p-PPARγ
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The extracted protein (25 μg) was separated electrophoretically in 4–15% polyacrylamide gradient precast gels (Mini-PROTEAN TGX; Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto a PVDF membrane (GE Healthcare, Tokyo, Japan). The membrane was blocked with PVDF blocking reagent from Can Get Signal (Toyobo, Osaka, Japan), and subsequently incubated overnight at 4°C with anti-phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ) (Ser 112) (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibody. After being washed with Tris-buffered saline-Tween, the membrane was incubated for 1 h with anti-rabbit IgG secondary antibody (1:5000 dilution; GE Healthcare, Tokyo, Japan). The membranes were reprobed with anti-PPARγ (H-100) (1:200 dilution) and anti-rabbit IgG (1:5000 dilution). The bands were detected with an ECL Western Blotting Detection Reagent (GE Healthcare, Tokyo, Japan) and quantified using an Ez-Capture (ATTO, Tokyo, Japan).
4
Western Blot Analysis of Signaling Proteins
5
Dystrophin C-terminus Western Blot
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Dp116 was analyzed by Western blotting, as described [25 (link)]. Blots were incubated overnight with a 1:1000 dilution of rabbit polyclonal antibody directed against the C-terminal domain of human dystrophin (ab154168; Abcam, Cambridge, UK), followed by incubation with anti-rabbit IgG secondary antibody (GE Healthcare). As a loading control, membranes were incubated with a 1:4000 dilution of GAPDH antibody (2118S; Cell Signaling Technology Inc.), followed by incubation with anti-mouse IgG secondary antibody (GE Healthcare). Immunoreactive bands were detected with Immobilon Forte Western HRP Substrate (Merck Millipore, MA, USA).
6
Quantification of Cell-Type Specific Proteins in Brain Nuclei
7
Liver Protein Extraction and Western Blotting
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Total proteins were extracted from liver samples and prepared for Western blotting according to the manufacturer’s instructions (Minute Total Protein Extraction Kit; Invent Biotechnology, Inc., Plymouth, MN, USA). Protein concentrations were evaluated using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The samples were size-fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyvinylidene difluoride (PVDF) membranes were used to transfer the protein from gel to membrane. Afterwards, membranes were incubated with anti-phosphorylated PERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-α-tubulin (MBL Co., Nagoya, Japan) antibodies diluted in blocking buffer. Next, anti-rabbit IgG secondary antibody (GE Healthcare, Pittsburgh, PA, USA) was used for incubation of the membranes. An ECL Prime Western Blotting Detection Reagent Kit (GE Healthcare, USA) was utilized for visualization of the enhanced chemiluminescent (ECL) membranes and images were captured using an Image Quant LAS 500 (GE Healthcare, USA). The images were analyzed with the ImageJ software from the NIH.
8
Quantification of Cell-Type Specific Proteins in Brain Nuclei
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Pelleted neuronal and glial nuclei fractions containing 100,000 nuclei each obtained from FACS (see Purification of cell-type specific nuclei from mouse brain above) were resuspended in 1X LDS sample buffer (Invitrogen) supplemented with 1% SDS and DTT (200 mM, Sigma). Nuclei resuspensions were sonicated in a Bioruptor on high power in 30 s On/30 s Off cycles for a total of 10 cycles at 4 °C. Samples were then boiled for ten minutes.
20 μg of protein samples or complete lysate from 100,000 nuclei were separated on 4-12% Bolt Bis-tris precast denaturing gels (Invitrogen, USA) and transferred onto PVDF membranes. Membranes were probed with primary antibodies diluted in 5% milk-TBST solution overnight at 4 °C. Membranes were then washed and probed with horseradish-peroxidase conjugated anti-mouse (GE), anti-rabbit IgG secondary antibody (GE), or anti-goat IgG antibody (Jackson Immunoresearch, USA) for one hour at room temperature. Membranes were developed using enhanced chemiluminescence substrate (PerkinElmer, USA) and exposed on film. Exposed films were scanned, and protein bands were quantified using ImageJ Software (NIH, USA). Protein quantities were normalized using beta-Actin, Gapdh or β III Neuron specific tubulin. All values were plotted relative to control littermate sample. Primary antibodies used seeSupplementary Table 8 .
20 μg of protein samples or complete lysate from 100,000 nuclei were separated on 4-12% Bolt Bis-tris precast denaturing gels (Invitrogen, USA) and transferred onto PVDF membranes. Membranes were probed with primary antibodies diluted in 5% milk-TBST solution overnight at 4 °C. Membranes were then washed and probed with horseradish-peroxidase conjugated anti-mouse (GE), anti-rabbit IgG secondary antibody (GE), or anti-goat IgG antibody (Jackson Immunoresearch, USA) for one hour at room temperature. Membranes were developed using enhanced chemiluminescence substrate (PerkinElmer, USA) and exposed on film. Exposed films were scanned, and protein bands were quantified using ImageJ Software (NIH, USA). Protein quantities were normalized using beta-Actin, Gapdh or β III Neuron specific tubulin. All values were plotted relative to control littermate sample. Primary antibodies used see
9
Western Blot Analysis of TMEM24 in MIN6 Cells
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After three washes with phosphate-buffered saline (PBS), MIN6 cells were homogenized and lysed in RIPA buffer (50 mM Tris-HCl, pH=7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 150 mM EDTA), placed on ice for 30 min or agitated on a rotator at 4°C for 30 min. Cell lysates were mixed with 3× sample buffer (6% SDS, 15% 2-mercaptoethanol, 30% glycerol, 0.006% Bromophenol Blue and 0.15 M Tris-HCl) and boiled at 95°C for 10 min. Protein content was determined by a detergent-compatible protein assay (Bio-Rad, Hercules, CA, USA) and proteins were separated by SDS-PAGE (5–20%) and blotted onto PVDF membrane using semi-dry transfer. Membranes were blocked in 4% milk dissolved in Tris-buffered saline with 0.1% Tween 20. The following antibodies and dilutions were used: anti-TMEM24 (Bethyl Laboratories, A304-764, 1:1000, specificity confirmed by KD and KO in this study), anti-GAPDH (Cell Signaling Technology, 14C10, 1:1000), anti-rabbit-IgG secondary antibody (GE Healthcare, 1:10,000). Membranes were developed with the Odyssey Fc Imaging system (LI-COR Bioscience).
10
Immunoblotting Assay for Protein Detection
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Mouse monoclonal anti-α-tubulin (T6199) was purchased from Sigma-Aldrich. Rabbit polyclonal anti-p27 (N-20) (#sc-527) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HRP-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from GE Healthcare (Little Chalfont, UK).
For immunoblotting, cells were suspended in lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 1 mM EGTA, 1% Nonidet P-40, 10% glycerol, cOmplete ULTRA tablets mini EDTA-free [Roche, Basel, Switzerland] and PhosSTOP [Roche]) and vortexed for 15 s. Cell lysates were subjected to immunoblotting with the indicated antibodies. Immune complexes were detected using the NOVEX ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) on an ImageQuant LAS 4000mini (GE Healthcare).
For immunoblotting, cells were suspended in lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 1 mM EGTA, 1% Nonidet P-40, 10% glycerol, cOmplete ULTRA tablets mini EDTA-free [Roche, Basel, Switzerland] and PhosSTOP [Roche]) and vortexed for 15 s. Cell lysates were subjected to immunoblotting with the indicated antibodies. Immune complexes were detected using the NOVEX ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) on an ImageQuant LAS 4000mini (GE Healthcare).
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