For Western blot analysis, 4D6 were treated with lysis buffer [25 mM tris-HCL (pH7.5), 50 mM NaCl (Sigma-Aldrich), 0.1% NP-40 (Sigma-Aldrich), 0.1% SDS (Sigma-Aldrich), 0.5% sodium deoxycholate (Sigma-Aldrich), 10% glycerol (Sigma-Aldrich), and protease inhibitors (1 tablet/10 ml; Roche)] followed by SDS–polyacrylamide gel electrophoresis and immunoblotting, using mouse anti-FLAG (monoclonal M2, Merck), rabbit anti-myc (Cell Signaling Technology), and rabbit anti–glyceraldehyde phosphate dehydrogenase (GAPDH) (Cell Signaling Technology) antibodies. For the relative quantification of FOXN1 in Fig. 1C , the loading control was run through the gel from the same loading well as the experimental sample. After the protein transfer, the membrane was cut in two parts at about 50 kDa, and each part was developed separately with anti-FLAG (top part) and anti-GAPDH (bottom part). For the relative quantification of FOXN1 protein levels, the fluorescence intensity of WT or Δ550 mutant FOXN1 protein bands were calculated relative to the fluorescence intensity of the GAPDH loading control after subtracting background fluorescence.
Rabbit anti glyceraldehyde phosphate dehydrogenase gapdh
Manufactured by Cell Signaling Technology
Rabbit anti-glyceraldehyde phosphate dehydrogenase (GAPDH) is a primary antibody that specifically recognizes GAPDH, an enzyme involved in glycolysis. This antibody can be used for the detection and quantification of GAPDH protein in various samples, such as cell lysates, tissue extracts, or purified protein preparations, using techniques like Western blotting, immunohistochemistry, or ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Sodium deoxycholate, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
Rabbit anti myc, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti flag m2, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Rabbit anti cleaved caspase 3, by Affinity Biosciences (1 mentions)
Rabbit anti α smooth muscle actin α sma, by Abcam (1 mentions)
Rabbit anti collagen 1, by Affinity Biosciences (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse igg, by Cell Signaling Technology (1 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
3 protocols using rabbit anti glyceraldehyde phosphate dehydrogenase gapdh
1
Western Blot Analysis of FOXN1 Mutants
2
Western Blot Analysis of Liver Proteins
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The liver tissues and LX2 cells were washed, homogenized on ice with RIPA buffer (Sigma-Aldrich, Saint Louis, MO, USA), and centrifuged at 8000 rpm for 10 min. The protein concentration in the supernatant was determined using a Bradford assay. Lysates containing equal amounts of protein were separated by SDS-PAGE. The western blot analysis was performed as previously described21 (link). The following primary antibodies were used in this study: rabbit anti-BCL2L10 (1:500, Origene), rabbit anti-glyceraldehyde phosphate dehydrogenase (GAPDH; 1:8000, Cell Signaling Technology), rabbit anti-cleaved caspase3 (1:400, Affinity), rabbit anti-cleaved caspase9 (1:400, Affinity), rabbit anti-collagen I (1:700, Affinity), rabbit anti-α-smooth muscle actin (α-SMA; 1:1000, Abcam), rabbit anti-TOM20 (1:2000, Affinity), and mouse anti-BCL2L10 (1:400, Abcam). After incubating with fluorescence-conjugated secondary antibodies (1:20000, Rockland Biochemicals), the immunoreactive bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences). For the protein quantification, the bands were scanned and quantified using Image-Pro plus 6.0 software (Datacell, UK), and GAPDH served as an internal control.
3
Western Blot Analysis of Signaling Proteins
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Cells were lyzed in Laemmli Sample Buffer (Bio‐Rad Laboratories, Hercules, CA), boiled for 5 min, and centrifuged at 4,000 rpm for 5 min. The samples were loaded on a 4–12% SDS–polyacrylamide gel, transferred to an Immobilon polyvinyl difluoride (PVDF) transfer membrane (Merck Millipore, Billerica, MA) and probed with primary antibodies followed by horseradish peroxidase (HRP)‐conjugated secondary antibody. Proteins were visualized with ECL Plus reagent (GE Healthcare, Chicago, IL), and the chemical luminescent reaction visualized using a FOTO/Analyst Luminary/Fx CCD imaging system (Fotodyne, Inc., Hartland, WI). The following antibodies were used in this study: Rabbit anti‐mTOR, rabbit anti‐phospho‐mTOR (p‐mTOR), rabbit anti‐phospho‐4E‐BP1 (p‐4E‐BP1) (Thr37/46), rabbit anti‐phospho‐p70‐S6 (p‐p70‐S6) kinase (Thr389), rabbit anti‐LC3, rabbit anti‐poly (ADP‐ribose) polymerase (PARP), rabbit anti‐p16, rabbit anti‐p21, rabbit anti‐glyceraldehyde phosphate dehydrogenase (GAPDH), HRP‐conjugated goat anti‐rabbit IgG, and HRP‐conjugated goat anti‐mouse IgG (all antibodies were from Cell Signaling Technology). The intensities of the bands were quantified using image analysis software, ImageJ version (NIH Image). Values were normalized relative to GAPDH expressions.
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