GAPDH Antibody (13H12) - BSA Free

Novus Biologicals | Catalog # NBP2-27103

Novus Biologicals

Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Human, Mouse, Rat, Drosophila, Monkey, Primate, Sheep

Cited:

Human, Mouse, Rat, Insect - Drosophila, Ovine, Primate, Primate - Macaca mulatta (Rhesus Macaque)

Predicted:

Bovine (91%), Canine (91%), Ferret (91%), Guinea Pig (91%), Porcine (91%), Reptile (91%), Squirrel (91%). Backed by our 100% Guarantee.

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence, Simple Western

Cited:

Western Blot, IF/IHC

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 kappa Clone # 13H12

Format

BSA Free
View all GAPDH Primary Antibodies »

Product Specifications

Immunogen

Amino acids between 275 and 325 of glyceraldehyde 3-phosphate dehydrogenase protein were used as the immunogen for this GAPDH antibody.

Reactivity Notes

Based upon 91% sequence similarity with immunogen, this antibody is predicted to react with Guinea Pig, Sheep, Squirrel, Porcine/Pig, Ferret, Canine/Dog/Cat, Bovine, Reptile / Rattlesnake and several other species. Immunogen shows 82% similarity to Xenopus and Zebrafish. Rat, sheep, and monkey reactivity reported in scientific literature (PMID: 24796753, PMID: 27618403, and PMID: 24462973 respectively).

Localization

Cytoplasm, Nucleus

Marker

Cytosolic Marker

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1 kappa

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.

Description

Novus Biologicals Mouse GAPDH Antibody (13H12) - BSA Free (NBP2-27103) is a monoclonal antibody validated for use in IHC, WB, ICC/IF and Simple Western. Anti-GAPDH Antibody: Cited in 35 publications. All Novus Biologicals antibodies are covered by our 100% guarantee.

Scientific Data Images for GAPDH Antibody (13H12) - BSA Free

Immunocytochemistry/ Immunofluorescence: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunocytochemistry/ Immunofluorescence: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunocytochemistry/Immunofluorescence: GAPDH Antibody (13H12) [NBP2-27103] - GAPDH antibody was tested in HeLa cells with Dylight 488 (green). Nuclei and alpha-tubulin were counterstained with DAPI (blue) and Dylight 550 (red). A dilution of 1:10 was used. Image objective 40x.
Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12) [NBP2-27103] - WB detection of GAPDH protein (theoretical molecular weight: 36 kDa) in HeLa cells lysate using GAPDH antibody (clone 13H12) in (A) the absence and (B) the presence of immunizing peptide.
Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) [NBP2-27103] - IHC-P detection GAPDH protein in a formalin-fixed paraffin-embedded section of human rectal carcinoma tissue using GAPDH antibody (clone 13H12) at 5 ug/ml concentration.
Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) [NBP2-27103] - IHC-P detection GAPDH protein in a formalin-fixed paraffin-embedded section of normal human breast tissue using GAPDH antibody (clone 13H12) at 5 ug/ml concentration.
Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12) [NBP2-27103] - Characterization of lncR492.(B) Northern blot of lncR494 using increasing amounts of loaded total. Black arrow signifies lncR492-specific signal at approximately 1400 bp. A probe targeting Gapdh mRNA served as the loading control. (D) RT-PCR analysis of lncR492 and Gapdh expression. RNA extract was treated with a 5'-phosphate-dependent exonuclease, resulting in a degradation of f.ex. ribosomal RNA.Citation: Winzi M, Casas Vila N, Paszkowski-Rogacz M, Ding L, Noack S, Theis M, et al. (2018) The long noncoding RNA lncR492 inhibits neural differentiation of murine embryonic stem cells. PLoS ONE 13(1): e0191682. https://doi.org/10.1371/journal.pone.0191682

Western Blot: GAPDH Antibody (13H12) [NBP2-27103] -

GAPDH-Antibody-13H12-Western-Blot-NBP2-27103-img0018.jpg
Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12) [NBP2-27103] - WB detection of GAPDH protein (theoretical molecular weight 36 kDa) in lysates of Mouse cell lines (A) NIH 3T3 (B) RAW 264.7 using GAPDH antibody (clone 13H12) at a concentration of 0.25 ug/ml.
Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Western Blot: GAPDH Antibody (13H12) [NBP2-27103] - analysis of GAPDH in HeLa and HEK 293 cells (25ug/lane) using anti-GAPDH antibody. Image from verified customer review.
Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) [NBP2-27103] - IHC-P detection GAPDH protein in a formalin-fixed paraffin-embedded section of normal human stomach tissue using GAPDH antibody (clone 13H12) at 5 ug/ml concentration.
Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) [NBP2-27103] - IHC-P detection GAPDH protein in a formalin-fixed paraffin-embedded section of human colon tissue using GAPDH antibody (clone 13H12) at 5 ug/ml concentration.
Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) - BSA Free [NBP2-27103]

Immunohistochemistry-Paraffin: GAPDH Antibody (13H12) [NBP2-27103] - IHC-P detection GAPDH protein in a formalin-fixed paraffin-embedded tissue section of human esophageal squamous cell carcinoma (SCC) using GAPDH antibody (clone 13H12) at 5 ug/ml concentration.
Simple Western: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Simple Western: GAPDH Antibody (13H12)BSA Free [NBP2-27103]

Simple Western: GAPDH Antibody (13H12) [NBP2-27103] - GAPDH/G3PDH Antibody (13H12) [NBP2-27103] - Simple Western lane view shows a specific band for GAPDH in 0.1 mg/ml of HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
GAPDH Antibody (13H12) - BSA Free

Western Blot: GAPDH Antibody (13H12) - BSA Free [NBP2-27103] -

Western Blot: GAPDH Antibody (13H12) - BSA Free [NBP2-27103] - Effect of AAT on Mcl-1 phosphorylation, the activity of MAP kinases & caspases.Neutrophils from healthy volunteers (2.5 × 106/ml) were cultured in medium supplemented with patient serum (3 mg protein/ml; 1%) & those containing low levels of AAT (AAT-reduce serum; 1%) in the presence of STS (0.2 μM). After 3 h, the expression of pMcl-1 (A, n = 7), pAkt (B, n = 8) & pERK1/2 (C, n = 10) were analyzed by western blot. Expression levels of the phosphorylated proteins were normalized to that of the unphosphorylated forms. GAPDH was used as loading control. One representative blot is displayed. *p<0.05; **p<0.01; n.s. = not significant. D. After 4 h of incubation the activities of caspase-9 & caspase-3/-7 were quantified. Results are presented as means ± SEM of eight independent experiments. No significant differences were found (one-way ANOVA with Newman keuls post-hoc test). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28493974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
GAPDH Antibody (13H12) - BSA Free

Western Blot: GAPDH Antibody (13H12) - BSA Free [NBP2-27103] -

Western Blot: GAPDH Antibody (13H12) - BSA Free [NBP2-27103] - Effect of AAT on Mcl-1 phosphorylation, the activity of MAP kinases & caspases.Neutrophils from healthy volunteers (2.5 × 106/ml) were cultured in medium supplemented with patient serum (3 mg protein/ml; 1%) & those containing low levels of AAT (AAT-reduce serum; 1%) in the presence of STS (0.2 μM). After 3 h, the expression of pMcl-1 (A, n = 7), pAkt (B, n = 8) & pERK1/2 (C, n = 10) were analyzed by western blot. Expression levels of the phosphorylated proteins were normalized to that of the unphosphorylated forms. GAPDH was used as loading control. One representative blot is displayed. *p<0.05; **p<0.01; n.s. = not significant. D. After 4 h of incubation the activities of caspase-9 & caspase-3/-7 were quantified. Results are presented as means ± SEM of eight independent experiments. No significant differences were found (one-way ANOVA with Newman keuls post-hoc test). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28493974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
GAPDH Antibody (13H12) - BSA Free

Western Blot: GAPDH Antibody (13H12) - BSA Free [NBP2-27103] -

Western Blot: GAPDH Antibody (13H12) - BSA Free [NBP2-27103] - Effect of AAT on Mcl-1 phosphorylation, the activity of MAP kinases & caspases.Neutrophils from healthy volunteers (2.5 × 106/ml) were cultured in medium supplemented with patient serum (3 mg protein/ml; 1%) & those containing low levels of AAT (AAT-reduce serum; 1%) in the presence of STS (0.2 μM). After 3 h, the expression of pMcl-1 (A, n = 7), pAkt (B, n = 8) & pERK1/2 (C, n = 10) were analyzed by western blot. Expression levels of the phosphorylated proteins were normalized to that of the unphosphorylated forms. GAPDH was used as loading control. One representative blot is displayed. *p<0.05; **p<0.01; n.s. = not significant. D. After 4 h of incubation the activities of caspase-9 & caspase-3/-7 were quantified. Results are presented as means ± SEM of eight independent experiments. No significant differences were found (one-way ANOVA with Newman keuls post-hoc test). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28493974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for GAPDH Antibody (13H12) - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:10

Immunohistochemistry

5 ug/ml

Immunohistochemistry-Paraffin

5 ug/ml

Simple Western

1:25

Western Blot

0.25 - 1 ug/ml
Application Notes

GAPDH is a widely used loading control for quantitative Western blotting. In IHC-P, the staining of formalin-fixed tissues is enhanced by boiling tissue sections in 10 mM sodium citrate buffer, pH 6.0 for 10-20 min followed by cooling at RT for 20 min.

In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in HeLa lysate 0.1 mg/mL, separated by Size, antibody dilution of 1:25, apparent MW was 44 kDa. Separated by Size-Wes, Sally Sue/Peggy Sue. WB reported in a verified customer review.

Reviewed Applications

Read 3 reviews rated 5 using NBP2-27103 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: GAPDH

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

Long Name

Glyceraldehyde-3-phosphate Dehydrogenase

Alternate Names

G3PDH

Entrez Gene IDs

2597 (Human)

Gene Symbol

GAPDH

UniProt

P04406 (Human)

Additional GAPDH Products

Product Documents for GAPDH Antibody (13H12) - BSA Free

Certificate of Analysis

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Product Specific Notices for GAPDH Antibody (13H12) - BSA Free

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.

Citations for GAPDH Antibody (13H12) - BSA Free

Customer Reviews for GAPDH Antibody (13H12) - BSA Free (3)

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Customer Images

Showing  1 - 3 of 3 reviews Showing All
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  • Name: Michaela Rekow
    Application: Western Blot
    Sample Tested: Splenocytes
    Species: Mouse
    Verified Customer | Posted 01/14/2016
  • Name: Anonymous
    Application: Western Blot
    Sample Tested: See PMID 24465661
    Species: Other
    Verified Customer | Posted 12/29/2014
  • Name: Anonymous
    Application: Western Blot
    Sample Tested: HeLa and HEK 293 cells
    Species: Human
    Verified Customer | Posted 10/09/2014
    Western blot for GAPDH
    GAPDH Antibody (13H12) - BSA Free NBP2-27103

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Protocols

View specific protocols for GAPDH Antibody (13H12) - BSA Free (NBP2-27103):

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 4% paraformaldehyde to the dish and fix at room temperature for 10 minutes.
2. Remove the paraformaldehyde and wash the cells in PBS.
3. Permeabilize the cells with 0.1% Triton X100 or other suitable detergent for 2 min.
4. Remove the permeabilization buffer and wash three times for 5 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 5 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 5 minutes each.
10. Counter stain DNA with DAPI if required.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer at all times).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.


Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for GAPDH Antibody (13H12) - BSA Free

Showing  1 - 5 of 10 FAQs Showing All

    • A: We offer sample sizes for many of our GAPDH loading control antibodies including NB300-221.

    • Q: I have a question about NBP2-27103 GAPDH ab for Simple Western Wes system. In the application note "In Simple Western only 10 - 15 uL of the recommended dilution is used per data point." What do you mean 10-15 micro L of dilution?
      A: It means to use only 10-15 ul of the diluted stock. Dilute from 0.25- 1 ug/ml then use 10- 15 ul on the column.

    • Q: I have GAPDH Antibody (NBP2-27103). According to the datasheet it is monoclonal. I want to do IF staining. Can it be stained with goat anti-rabbit IgG-FITC.
      A: Our GAPDH antibody with catalogue number NBP2-27103 has been validated for immunofluorescence. Our own immunofluorescence testing data was generated in HeLa cells; detection was with a Dylight 488-conjugated anti-mouse secondary antibody. Since NBP2-27103 is a mouse antibody you will need to use an anti-mouse secondary antibody for detection; a goat anti-rabbit antibody will not be suitable. NBP2-27103 is of the isotype IgG1 kappa, and you can see our anti-mouse IgG1 secondary antibodies at the following link: anti-mouse IgG1 secondary antibodies You can use the search filters at the left hand side of our webpage to select your conjugate of choice.

    • Q: I need a suitable loading control antibody that I can use in western blot of rat nerve extracts, which do you recommend?
      A: 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.

    • A: 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.

    • Q: 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.
      A: For homogenized tissue, beta-actin and GAPDH are both fine.

    • Q: 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?
      A: 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.

    • Q: We need a loading control antibody for WB working with HeLa cell line, will this GAPDH antibody work for that?
      A: 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.

    • Q: We received GAPDH antibody NB600-502 and there was only about 15µl of antibody in the tube. I was under the impression this should have been 100µl.
      A: The unit size of this product is 0.1 mg with a concentration of 6.7 mg/ml. Hence the volume should be no less than 14.9 ul. 

    • A: This GAPDH antibody is raised in mouse so you would want to use an anti-mouse secondary with the dye of your choice.
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