Standard Western blotting method from our published protocol was used with minor modifications (Kamiya et al., 2005 ; Kamiya et al., 2006 (link)). Protein samples were analyzed by SDS-PAGE and Western blotting with the following antibodies: rabbit anti-DISC1 polyclonal antibody (1:500, ECM Biosciences), mouse anti-GAPDH monoclonal antibody (1:10,000, Santa Cruz), mouse anti-β-tubulin monoclonal antibody (1:5000, Sigma-Aldrich). For co-immunoprecipitation, we used rabbit polyclonal anti-HA (10ug, Clontech) and mouse monoclonal anti-myc (5ug, SantaCruz) antibodies. Species-appropriate secondary antibodies were conjugated to HRP (Pierce) and detected by enhanced chemiluminescence (Thermo Scientific) using ImageQuant LAS4000 mini (GE Healthcare). Quantification of immunoblot was performed with ImageJ64 (National Institutes of Health). Optical density of immunoreactivity in Western blotting was acquired with Image J software (http://rsb.info.nih.gov/ij/ ). Protein levels were normalized to GAPDH and expressed as relative % integrated intensity.
Anti ha
Manufactured by Takara Bio
Sourced in United States
The Anti-HA product is a laboratory reagent used to detect and purify proteins that have been tagged with the hemagglutinin (HA) epitope. It provides a specific and reliable method for identifying and isolating HA-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho akt s473, by Cell Signaling Technology (3 mentions)
Anti myc, by Takara Bio (3 mentions)
Anti gst, by Santa Cruz Biotechnology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Nonfat dry milk, by HiMedia (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Mouse anti gapdh monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti β tubulin antibody, by Merck Group (1 mentions)
Mouse monoclonal anti myc, by Santa Cruz Biotechnology (1 mentions)
Anti mdm2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Hrp linked goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti actin, by Abcam (1 mentions)
Anti nedd4, by Merck Group (1 mentions)
Goat anti mouse igg hrp conjugate, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti myc, by Developmental Studies Hybridoma Bank (1 mentions)
Ab140601, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
6 protocols using anti ha
1
Western Blotting Analysis of DISC1 Protein
2
Protein Expression and Western Blot Analysis
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Human embryonic kidney 293T cells were transfected with gene of interest for 24 h. The cells were harvested and lysed in RIPA lysis buffer (1% NP-40, 20 mM TrisCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% sodium deoxycholate, 1 mM Na3VO4). Protein estimation was carried out using BCA Protein Assay Kit (Pierce, Thermo Scientific, United States). An equal amount of protein was loaded on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and was transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk (Himedia Laboratories, India). The primary antibodies used were anti-AKT, anti-Mdm2, anti-AKT substrate, anti-GAPDH, anti–phospho-AKT (S473) (Cell Signaling Technology), anti-Myc, anti-HA (Clontech), anti-GST (Santa Cruz Biotechnology), and anti-Vif (NIH, MD, United States). The secondary antibodies used were anti-rabbit/mouse–horseradish peroxidase–conjugated (Jackson ImmunoResearch). Blots were developed using ECL (enhanced chemiluminescence) reagent.
3
Western Blot Antibody Detection Protocol
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Western blotting was performed with anti-myc (Developmental Studies Hybridoma Bank at the University of Iowa, deposited by Dr. J. Michael Bishop, catalog no. 9E 10), anti-HA (Clontech, catalog no. 631207), anti-hIFITM3 (Proteintech Group, catalog no. 11714-1-AP), anti-mIFITM3 (Abcam, catalog no. ab65183), anti-NEDD4 (Millipore, catalog no. 07–049), anti-FLAG (Sigma, catalog no. F7425), anti-actin (Abcam, catalog no. ab3280), or anti-GAPDH (Invitrogen, catalog no. 398600) antibodies. All primary antibodies were used at a 1:1000 dilution. Secondary antibodies, Goat Anti-Mouse IgG, HRP conjugate (Millipore catalog no. 12–349), Goat Anti-Rabbit IgG, HRP-linked (Cell Signaling, catalog no. 70745), and Goat Anti-Mouse, IgG1 Gamma 1 Heavy Chain Specific (SouthernBiotech, catalog no. 1070–05, specifically used for detecting immunoprecipitated protein ubiquitination) were all diluted at 1:20,000.
4
Analyzing Protein Expression in HEK-293T Cells
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HEK-293T cells were transfected with the gene of interest for either 24 h or 36 h. The cells were harvested and lysed in radioimmunoprecipitation assay lysis buffer (1% NP-40, 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% sodium deoxycholate, and 1 mM Na3VO4). Protein estimation was carried out by using BCA Protein Assay Kit (Pierce, Thermo Scientific). An equal amount of protein was loaded on SDS-PAGE and transferred to nitrocellulose membrane as described before (24 (link)). The membranes were blocked with 5% nonfat dry milk (Himedia Laboratories) or 5% bovine serum albumin. The primary antibodies used were anti-AKT, anti-GAPDH, anti-phospho-AKT (S-473) (Cell Signaling Technology), anti-Myc, anti-HA (Clontech), ab140601 (abcam–K48 antibody), and anti-GST (Santa Cruz Biotechnology). The secondary antibodies used were anti-rabbit/mouse-horseradish peroxidase conjugated (Jackson ImmunoResearch). Blots were developed using Enhanced Chemiluminescence) reagent.
5
Subcellular Localization of HA-KLF1-ER^T2 in Macrophages
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To assess HA-KLF1-ERT2 sub-cellular localization, 6 × 104 macrophages were cultured in a gelatin-coated Nunc® Lab-Tek® Chamber Slide System (Sigma), fixed in 4% PFA in PBS at room temperature for 10 min, permeabilized in PBS-T (PBS with 0.4% Triton-X100) for 20 min and blocked in PBS-T with 1% BSA and 3% goat serum for 2 h and incubated in anti-HA 1:500 (Clontech #631207) for 1.5 h. Cells were washed with PBS-T thrice for 15 min, incubated in Alexa488 anti-rabbit 1:1000 (Thermo Fisher Scientific #A-11008) for 1.5 h in the dark, washed with PBS-T thrice for 15 min then counter-stained with DAPI 1:1000 (Sigma) for 5 min.
6
Protein Analysis of Transfected HEK-293T Cells
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HEK-293T cells were transfected with gene of interest for 36 hrs. The the cells were harvested and lysed in RIPA lysis buffer (1% NP-40, 20mM TrisCl, pH 7.5, 150 mM NaCl, 1mM Na2EDTA, 1mM EGTA, 1% Sodium deoxycholate, 1mM Na3VO4). Protein estimation was carried out using BCA Protein Assay Kit (Pierce, Thermo Scientific, USA).
An equal amount of protein was loaded on SDS-PAGE and was transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk (Himedia Laboratories, India). The primary antibodies used were anti-AKT, anti-GAPDH, anti-phospho-AKT (S473) (Cell Signaling Technology), anti-myc, anti-HA (Clontech), anti-GST (Santa Cruz Biotechnology). The secondary antibodies used were antirabbit/mouse-HRP conjugated (Jackson Immuno Research). Blots were developed using ECL (Enhanced Chemiluminescence) reagent.
An equal amount of protein was loaded on SDS-PAGE and was transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk (Himedia Laboratories, India). The primary antibodies used were anti-AKT, anti-GAPDH, anti-phospho-AKT (S473) (Cell Signaling Technology), anti-myc, anti-HA (Clontech), anti-GST (Santa Cruz Biotechnology). The secondary antibodies used were antirabbit/mouse-HRP conjugated (Jackson Immuno Research). Blots were developed using ECL (Enhanced Chemiluminescence) reagent.
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