GAPDH Antibody (2D4A7)

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Product Details

Summary
Product Discontinued
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Order Details


    • Catalog Number
      NB300-328
    • Availability
      Product Discontinued

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GAPDH Antibody (2D4A7) Summary

Immunogen
GAPDH antibody raised against Ni-NTA purified recombinant human GAPDH expressed in E. Coli strain BL21 (DE3). [UniProt# P04406]
Marker
Cytosolic Marker
Isotype
IgG1 Kappa
Clonality
Monoclonal
Host
Mouse
Gene
GAPDH
Purity
Ammonium sulfate precipitation
Innovator's Reward
Test in a species/application not listed above to receive a full credit towards a future purchase.

Applications/Dilutions

Dilutions
  • ELISA 1:100-1:2000
  • Immunocytochemistry/Immunofluorescence 1:200-1:1000
  • Immunohistochemistry 1:1000-1:4000
  • Immunohistochemistry-Paraffin 1:1000-1:4000
  • Immunoprecipitation
  • Simple Western 1:250
  • Western Blot 1:20000
Application Notes
In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Separated by Size-Wes, Sally Sue/Peggy Sue.
The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. Use in immunoprecipitation reported in scientific literature (PMID: 25915564).
Theoretical MW
36 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Control
GAPDH Overexpression Lysate
Reviewed Applications
Read 12 Reviews rated 5
using
NB300-328 in the following applications:

Publications
Read Publications using
NB300-328 in the following applications:

  • 1 publication
  • 2 publications
  • IP
    1 publication
  • WB
    56 publications

Reactivity Notes

Since GAPDH is one of the most conserved proteins known it is therefore likely that the antibody will react with other species. Porcine reactivity reported in scientific literature (PMID: 25063310)

Packaging, Storage & Formulations

Storage
Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
Buffer
PBS
Preservative
0.03% Sodium Azide
Concentration
1.0 mg/ml
Purity
Ammonium sulfate precipitation

Alternate Names for GAPDH Antibody (2D4A7)

  • aging-associated gene 9 protein
  • EC 1.2.1
  • EC 1.2.1.12
  • EC 2.6.99.-
  • G3PD
  • G3PDH
  • GAPD
  • GAPDH
  • glyceraldehyde 3-phosphate dehydrogenase
  • glyceraldehyde-3-phosphate dehydrogenase
  • MGC88685
  • Peptidyl-cysteine S-nitrosylase GAPDH

Background

Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis, converting glyceraldehyde-3-phosphate into 1,3 diphosphoglycerate. This constitutively expressed, homotetramer protein can be found in the nucleus and cytoplasm, and the monomer has a theoretical molecular weight of 36 kDa. Known as a housekeeping gene, GAPDH routinely serves as a control for real-time PCR (RT-PCR) and as a loading control for Western Blots (1-3). In addition to its role in glycolysis, GAPDH has multiple cellular functions including membrane trafficking, transcription activation, apoptosis, DNA repair, and has been implicated in various pathologies such as cancer and neurodegenerative diseases including Alzheimer's and Huntington's (4,5).

References

1) Barber RD, Harmer DW, Coleman RA, Clark BJ. (2005) GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics. 21(3):389-95. PMID: 15769908

2) Jia Y, Takimoto K. (2006) Mitogen-activated protein kinases control cardiac KChIP2 gene expression. Circ Res. 98(3):386-93. PMID: 16385079

3) Godsel LM, Hsieh SN, Amargo EV, Bass AE, Pascoe-McGillicuddy LT, Huen AC, Thorne ME, Gaudry CA, Park JK, Myung K, Goldman RD, Chew TL, Green KJ. (2005) Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin. J Cell Biol. 171(6):1045-59. PMID: 16365169

4) Sirover MA1. (1999) New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta. 1432(2): 159-84. PMID: 10407139

5) Tristan C, Shahani N, Sedlak TW, Sawa A. (2011) The diverse functions of GAPDH: views from different subcellular compartments. Cell Signal. 23(2):317-23. PMID: 20727968

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Publications for GAPDH Antibody (NB300-328)(65)

We have publications tested in 5 confirmed species: Human, Mouse, Rat, Porcine, Rabbit.

We have publications tested in 4 applications: Flow, ICC/IF, IP, WB.


Filter By Application
Flow
(1)
ICC/IF
(2)
IP
(1)
WB
(56)
All Applications
Filter By Species
Human
(46)
Mouse
(4)
Rat
(2)
Porcine
(2)
Rabbit
(1)
All Species
Showing Publications 1 - 10 of 65. Show All 65 Publications.
Publications using NB300-328 Applications Species
Sharma V, Thakore P, Majumdar S THAP9 Transposase Cleaves DNA via Conserved Acidic Residues in an RNaseH-Like Domain Cells May 29 2021 [PMID: 34072453] (WB) WB
Camporesi E, Lashley T, Gobom J et al. Neuroligin-1 in brain and CSF of neurodegenerative disorders: investigation for synaptic biomarkers Acta neuropathologica communications Feb 1 2021 [PMID: 33522967] (WB, Human) WB Human
Unsicker C, Cristian FB, von Hahn M, et al. SHANK2 mutations impair apoptosis, proliferation and neurite outgrowth during early neuronal differentiation in SH-SY5Y cells Scientific reports Jan 22 2021 [PMID: 33483523] (WB, Human) WB Human
Hung WT, Wang CH, Lin SY et al. Leptin protects brain from ischemia/reperfusion-induced infarction by stabilizing the blood-brain barrier to block brain infiltration of the blood-borne neutrophils Eur. J. Neurosci. Jul 7 2020 [PMID: 32638449]
Eriksson I, WAster P, Ollinger K Restoration of lysosomal function after damage is accompanied by recycling of lysosomal membrane proteins Cell Death Dis May 14 2020 [PMID: 32409651] (WB, Human) WB Human
Lachenauer, E R, Stabler, S P Et al. p53 Disruption Increases Uracil Accumulation in DNA of Murine Embryonic Fibroblasts and Leads to Folic Acid-Nonresponsive Neural Tube Defects in Mice. J Nutr Jul 1 2020 [PMID: 32271909] (Flow, Human) Flow Human
Wang S, Lin M, Hsu F et al. Synthesis and Biological Evaluation of 2-Quinolineacrylamides Bioorganic & Medicinal Chemistry Dec 1 2019 [PMID: 31924504] (WB, Human) WB Human
Cvitic S, Strutz J, Appel HM Sexual dimorphism of miRNA signatures in feto-placental endothelial cells is associated with altered barrier function and actin organisation Clin. Sci. Dec 11 2019 [PMID: 31825070]
Shea J, Terry C, Edwards K, Agarwal J Glitazone loaded fat enhances adiponectin production and inhibits breast cancer cell proliferation Mol. Biol. Rep. 2019 Sep 30 [PMID: 31571108] (WB, Human) WB Human
Zhang Q, Narayanan V, Mui KL et al. Mechanical Stabilization of the Glandular Acinus by Linker of Nucleoskeleton and Cytoskeleton Complex Curr. Biol. Aug 2 2019 [PMID: 31402305]
Show All 65 Publications.

Reviews for GAPDH Antibody (NB300-328) (12) 512

Average Rating: 5
(Based on 12 reviews)
We have 12 reviews tested in 4 species: Human, Mouse, Human and Mouse, Pig.

Reviews using NB300-328:
Filter by Applications
WB
(9)
ICC
(3)
All Applications
Filter by Species
Human
(9)
Mouse
(1)
Human and Mouse
(1)
Pig
(1)
All Species
Images Ratings Applications Species Date Details
Western Blot GAPDH NB300-328
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5
reviewed by:
Verified Customer
WB Human 04/04/2020
View

Summary

ApplicationWestern Blot
Sample Tested hela cell,A2780 ovarian cancer cells
SpeciesHuman
LotE-3
Western Blot GAPDH NB300-328
Enlarge
5
reviewed by:
Verified Customer
WB Human 12/27/2019
View

Summary

ApplicationWestern Blot
Sample TestedA549 cells
SpeciesHuman
LotE-3
Western Blot GAPDH NB300-328
Enlarge
5
reviewed by:
Verified Customer
WB Human and Mouse 02/19/2019
View

Summary

ApplicationWestern Blot
Sample TestedB16-F1 mouse melanoma cell line,HCC827 human non-small cell lung cancer cell line
SpeciesHuman and Mouse
LotE-3
  5
reviewed by:
Verified Customer
WB Human 01/31/2019
View

Summary

ApplicationWestern Blot
Sample TestedBreast cancer cells
SpeciesHuman
LotE-1
Western Blot GAPDH NB300-328
Enlarge
5
reviewed by:
Verified Customer
WB Mouse 10/28/2018
View

Summary

ApplicationWestern Blot
Sample Testedct26,EMT6 cells
SpeciesMouse
LotE-3
Western Blot GAPDH NB300-328
Enlarge
5
reviewed by:
Verified Customer
WB Human 07/17/2018
View

Summary

ApplicationWestern Blot
Sample TestedBreast cancer cells
SpeciesHuman
LotE-1

Comments

CommentsTotal cell lysates from T47D, MDA-MB-231, MCF-7 and MCF-10A cells were subjected to western blot. PVDF membrane were probed with 0.2 mm/ml Human GAPDH Antibody (2D4A7) (NB300-328). A specific band was detected for GAPDH at approximately 37 kDa. This experiment was conducted under reducing conditions
Immunocytochemistry GAPDH NB300-328
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5
reviewed by:
Verified Customer
ICC Pig 01/04/2017
View

Summary

ApplicationImmunocytochemistry
Sample TestedPig TM cells
SpeciesPig

Comments

CommentsLabeling of GAPDH in pig TM cells using GAPDH antibody (2D4A7) at 1: 1000 dilution and Alexa 594 (red)
  5
reviewed by:
Verified Customer
WB Human 09/19/2014
View

Summary

ApplicationWestern Blot
Sample TestedGAPDH immunoblot in MCF7 cells
SpeciesHuman

Blocking

Blocking DetailsNon-fat dry milk (5%) in PBS

Primary Anitbody

Dilution Ratio1:2000 in PBS at RT for 3 hours

Secondary Antibody

Secondary DescriptionAnti mouse IgG (HRP-conjugated)
Secondary Manufacturer Cat#ab6789 (Abcam)
Secondary Concentration1:5K

Details

Detection NotesBio-Rad Immun-Star HRP Substrate was used with X-ray film, Exposure time: 5sec
Immunocytochemistry GAPDH NB300-328
Enlarge
5
reviewed by:
Bryan Tinsley
ICC Human 06/10/2014
View

Summary

ApplicationImmunocytochemistry
SpeciesHuman
Special ApplicationsIF Confocal analysis of Hela cells using GAPDH antibody (NB300-328, 1:50). An Alexa Fluor 488-conjugated Goat to mouse IgG was used as secondary antibody (green). Actin filaments were labeled with Alexa Fluor 568 phalloidin (red). DAPI was used to stain the cell nuclei (blue).
FileView PDF
Western Blot GAPDH NB300-328
Enlarge
5
reviewed by:
Bryan Tinsley
WB Human 06/10/2014
View

Summary

ApplicationWestern Blot
SpeciesHuman
Special ApplicationsWestern blot analysis of extracts from Jurkat and HeLa cells using GAPDH antibody (NB300-328, 1:1000).
FileView PDF
Immunocytochemistry GAPDH NB300-328
Enlarge
5
reviewed by:
Bryan Tinsley
ICC Human 05/21/2014
View

Summary

ApplicationImmunocytochemistry
SpeciesHuman
FileView PDF
Western Blot GAPDH NB300-328
Enlarge
5
reviewed by:
Mary Ndiaye
WB Human 06/10/2011
View

Summary

ApplicationWestern Blot
Sample TestedHaCaT whole cell lysate, Sample Amount: 30 ug
SpeciesHuman
LotB
CommentsThis antibody works great for western blotting with little to no background, even at very dilute concentrations (we did 1:20,000 successfully, and could probably go lower).

Blocking

Blocking DetailsBlocking Buffer: 5% milk in TBST, Blocking Time: 30 minutes, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioPrimary Ab Dilution Ratio: 1:20,000, Incubation Dilution Buffer: 5% Milk in TBST, Incubation Time: 2 hrs, Incubation Temp: RT

Secondary Antibody

Secondary DescriptionSecondary Ab: HRP-conjugated anti-mouse, Secondary Ab Dilution Ratio: 1:3000
Secondary Manufacturer Cat#Cell Signaling 7076

Details

Detection NotesDetection Method: Chemiluminescent, on Kodak Imager, Exposure Time:30sec, Pos Ctrl: Untreated cells, Specific Bands: 36 kDa

Comments

CommentsThis antibody works great for western blotting with little to no background, even at very dilute concentrations (we did 1:20,000 successfully, and could probably go lower).

Product General Protocols

View specific protocols for GAPDH Antibody (NB300-328): Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

Video Protocols

WB Video Protocol
ICC/IF Video Protocol

FAQs for GAPDH Antibody (NB300-328). (Showing 1 - 7 of 7 FAQs).

  1. We need a loading control antibody for WB working with HeLa cell line, will this GAPDH antibody work for that?
    • GAPDH is expressed in HeLa cell lines but whether or not it is the best option for a loading control antibody depends on the size of the protein you are interested in. GAPDH antibody is a good choice for low to mid MW targets, as GAPDH detects around 35 kDa.
  2. I want to know why GAPDH is used as a loading control antibody in WB techniques.
    • Our GAPDH antibody makes a good loading control because GAPDH is  a housekeeping gene essential to metabolism that is globally expressed in most tissue types and cell lines.
  3. I need a suitable loading control antibody that I can use in western blot of rat nerve extracts, which do you recommend?
    • This GAPDH antibody (NB300-221) is a good choice for a loading control antibody and has been cited in over 180 publications and used on rat lysates with good results.
  4. I'm looking for a loading control between 24kDa - 65kDa for use in simple western, we tried an actin antibody  we already had but it didn't work. What can you recommend?
    • Our GAPDH antibody NB300-221 has been validated in simple western and detects around 35 kDa so I think it would be a good choice for a simple western loading control antibody based on your size requirements.
  5. What secondary antibody should I use for visualization of the GAPDH antibody?
    • This GAPDH antibody is raised in mouse so you would want to use an anti-mouse secondary with the dye of your choice.
  6. Do you offer a smaller size for the GAPDH antibodies?
    • We offer sample sizes for many of our GAPDH loading control antibodies including NB300-221.
  7. I wanted to know which of the two housekeeping genes B-actin or GAPDH can be used for best results. I intend to do an experiment to determine expression of IL-6 and TNF-alpha in liver and kidney tissues.
    • For homogenized tissue, beta-actin and GAPDH are both fine.

Control Lysate(s)

Secondary Antibodies

 

Isotype Controls

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Blogs on GAPDH. Showing 1-10 of 13 blog posts - Show all blog posts.

Considerations for Quantitative Western blotting
By Jamshed Arslan, Pharm. D., PhD. Since its inception in 1979, Western blotting has undergone several developments. The use of radioactive probes was common throughout 1980s, but utilizing secondary antibodies labell...  Read full blog post.

The use of Beta Actin (AC-15) as a loading control across multiple species
Actin is a fundamental component of the cytoskeleton, where it has the ability to create and break down actin filament formation in response to various cell needs.  Actin has six highly conserved isoforms, however beta and gamma actin are the two...  Read full blog post.

Tips on choosing an ideal loading control antibody for Western Blotting
Western blotting is one of the most commonly used antibody assay techniques in cell and molecular biology research since its development over three decades ago, and is considered the gold standard for protein detection and quantification. When p...  Read full blog post.

GAPDH - A "housekeeping" gene with diverse functions in cellular homeostasis
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a well-known housekeeping gene with functions in glycolysis. Many biologists are familiar with the gene and use GAPDH antibodies for a loading control when performing western blots. However, this ...  Read full blog post.

A New Standard in Antibody Testing - Simple Western Certified Antibodies
The Western blot is one of the most commonly used antibody assay techniques in cell and molecular biology research since its development over three decades ago, and is considered the gold standard for protein detection and quantification. The tradi...  Read full blog post.

GAPDH: More than a housekeeping gene
GAPDH is a 146kD tetramer glycolytic pathway metabolic enzyme composed of four 30-40 kDa subunits. It is responsible for reversibly phosphorylating its substrate glyceraldehyde 3-phosphate within the glycolytic pathway.  Apart from its role in gl...  Read full blog post.

GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase)
GAPDH is a 146 kD tetramer glycolytic pathway metabolic enzyme responsible for reversibly phosphorylating glyceraldehyde 3-phosphate. It may have other possible functions in transcriptional activation. GAPDH is highly expressed due to this housekeepin...  Read full blog post.

Time to Start Actin Like a Reliable 'Housekeeper'!
A growing body of data and studies using actin antibodies supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properti...  Read full blog post.

Beta Actin and GAPDH: The Importance of Loading Controls
Western blotting is an essential technique to probe protein expression in complex cell or tissue lysates. To accurately determine protein expression and interpret Western blot results, it is important to use loading controls. A loading control anti...  Read full blog post.

The GAPDH Antibody in Western blot Assays
The loading controls on our antibody database are widely used in gel electrophoresis and Western blotting studies. Products like the GAPDH antibody detect "housekeeping" proteins which are abundantly distributed in cells. This makes them useful for ch...  Read full blog post.

Showing 1-10 of 13 blog posts - Show all blog posts.
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Recent Reviews

5
5
12
4
0
3
0
2
0
1
0

Verified Customer
04/04/2020
Application: WB
Species: Human

Verified Customer
12/27/2019
Application: WB
Species: Human

Verified Customer
02/19/2019
Application: WB
Species: Human and Mouse

Bioinformatics

Gene Symbol GAPDH
Entrez
Uniprot