Immunofluorescence analysis of GAPDH was performed using 70% confluent log phase NIH/3T3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with GAPDH Rabbit Polyclonal Antibody (PA116777) at 2µg/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse, Non-human primate, Rat|
|Published species reactivity||Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Residues between 150 and 200 of human Glyceraldehyde-3-Phosphate Dehydrogenase (GeneID 2597)|
|Purification||Antigen affinity chromatography|
|Storage buffer||TBS with 0.1% BSA|
|Contains||0.09% sodium azide|
|Storage Conditions||4° C, do not freeze|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:100-1:500|
|Western Blot (WB)||0.5 µg/mL|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 1 publications below|
Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended.
Suggested positive control: antigen standard for GAPDH (transient overexpression lysate).
GAPDH is a 146 kDa tetramer composed of four 30-40 kDa subunits. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) is a metabolic enzyme responsible for catalyzing one step in the glycolytic pathway, the reversible oxidative phosphorylation of glyceraldehyde 3-phosphate. Because GAPDH as a protein expressed in large amounts and which is required at all times for an important house keeping functions, levels of GAPDH mRNA are often used as standards in studies of mRNA expression. Increasingly, scientists are making use of specific antibodies to GAPDH as loading controls for western blotting experiments. Apart from a role in glycolysis, GAPDH may have other roles such as in the activation of transcription. GAPDH is reported to bind to a variety of other proteins, including the amyloid precursor protein, mutations in which cause some forms of Alzheimer's disease, and the polyglutamine tracts of Huntingtin, the protein product aberrant forms of which are causative of Huntington's disease. Associations with actin and tubulin have also be reported. The protein may also have a role in the regulation of apoptosis, and interestingly migrates from the cytoplasm into the nucleus when cells become apoptotic.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
PAFAH1B1 and the lncRNA NONHSAT073641 maintain an angiogenic phenotype in human endothelial cells.
PA1-16777 was used in western blot to analyze maintenance of an angiogenic phenotype in human endothelial cells by PAFAH1B1 and the IncRNA NONHSAT073641
|Josipovic I,Fork C,Preussner J,Prior KK,Iloska D,Vasconez AE,Labocha S,Angioni C,Thomas D,Ferreirós N,Looso M,Pullamsetti SS,Geisslinger G,Steinhilber D,Brandes RP,Leisegang MS||Acta physiologica (Oxford, England) (218:13)||2016|