Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

Product name

Anti-GAPDH antibody [6C5] - Loading Control
See all GAPDH primary antibodies
Description

Mouse monoclonal [6C5] to GAPDH - Loading Control
Host species

Mouse
Specificity

This antibody can be used as a loading control antibody, however we also recommend ab9484 as an alternative. GAPDH is a 146 kDa tetramer composed of four 30-40 kDa subunits. There is no cross-reaction with GAPDH from yeast. Preliminary data indicates that the antibody recognizes the monomer (36 kDa) and also the dimer forms of GAPDH, but not the tetrameric form of the protein.
Tested applications

Suitable for: ELISA, ICC, WB, ICC/IF, IHC-Frmore details
Species reactivity

Reacts with: Mouse, Rat, Rabbit, Chicken, Hamster, Cat, Dog, Human, Pig, Xenopus laevis, Fish, Monkey, Zebrafish, Baboon, African green monkey
Predicted to work with: Horse, Guinea pig, Xenopus tropicalisDoes not react with: Goat, Cow, Saccharomyces cerevisiae
Immunogen

Rabbit muscle GAPDH.
Positive control
- ICC/IF: HeLa cells, NIH3T3 cells, SV40LT-SMC cells
General notes

Abcam recommended secondaries - Goat Anti-Mouse HRP (ab205719) and Goat Anti-Mouse Alexa Fluor^® 488 (ab150113).
See other anti-mouse secondary antibodies that can be used with this antibody.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.40
Preservative: 0.09% Sodium azide
Constituent: PBS
Concentration information loading...
Purity

Protein A purified
Purification notes

Chromatography on protein A Sepharose
Clonality

Monoclonal
Clone number

6C5
Myeloma

Sp2/0
Isotype

IgG1
Research areas

Our Abpromise guarantee covers the use of ab8245 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
ELISA		Use at an assay dependent concentration.
ICC		Use at an assay dependent concentration.
WB		1/500 - 1/10000. Detects a band of approximately 36 kDa (predicted molecular weight: 40.2 kDa). PubMed: 16450009
ICC/IF		Use a concentration of 1 - 5 µg/ml.
IHC-Fr		1/500.

Function

Has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. Participates in nuclear events including transcription, RNA transport, DNA replication and apoptosis. Nuclear functions are probably due to the nitrosylase activity that mediates cysteine S-nitrosylation of nuclear target proteins such as SIRT1, HDAC2 and PRKDC (By similarity). Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in glycolysis that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate.
Pathway

Carbohydrate degradation; glycolysis; pyruvate from D-glyceraldehyde 3-phosphate: step 1/5.
Sequence similarities

Belongs to the glyceraldehyde-3-phosphate dehydrogenase family.
Post-translational
modifications

S-nitrosylation of Cys-152 leads to interaction with SIAH1, followed by translocation to the nucleus.
ISGylated.
Cellular localization

Cytoplasm > cytosol. Nucleus. Cytoplasm > perinuclear region. Membrane. Translocates to the nucleus following S-nitrosylation and interaction with SIAH1, which contains a nuclear localization signal (By similarity). Postnuclear and Perinuclear regions.
Target information above from: UniProt accession P04406 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 374193 Chicken
- Entrez Gene: 403755 Dog
- Entrez Gene: 2597 Human
- Entrez Gene: 100042025 Mouse
- Entrez Gene: 14433 Mouse
- Entrez Gene: 100009074 Rabbit
- Entrez Gene: 24383 Rat
- Entrez Gene: 685186 Rat
- Entrez Gene: 380259 Xenopus laevis
- Entrez Gene: 317743 Zebrafish
- Omim: 138400 Human
- SwissProt: P00356 Chicken
- SwissProt: Q28259 Dog
- SwissProt: P04406 Human
- SwissProt: P16858 Mouse
- SwissProt: P00355 Pig
- SwissProt: P46406 Rabbit
- SwissProt: P04797 Rat
- SwissProt: P51469 Xenopus laevis
- SwissProt: Q5XJ10 Zebrafish
- Unigene: 544577 Human
- Unigene: 592355 Human
- Unigene: 598320 Human
- Unigene: 304088 Mouse
- Unigene: 309092 Mouse
- Unigene: 317779 Mouse
- Unigene: 343110 Mouse
- Unigene: 392463 Mouse
- Unigene: 392480 Mouse
- Unigene: 414470 Mouse
- Unigene: 458138 Mouse
- Unigene: 458416 Mouse
- Unigene: 475698 Mouse
- Unigene: 129558 Rat
- Unigene: 91450 Rat
- Unigene: 995 Xenopus laevis
- Unigene: 35640 Zebrafish
see all
Alternative names
- 38 kDa BFA-dependent ADP-ribosylation substrate antibody
- aging associated gene 9 protein antibody
- Aging-associated gene 9 protein antibody
- BARS-38 antibody
- cb609 antibody
- EC 1.2.1.12 antibody
- Epididymis secretory sperm binding protein Li 162eP antibody
- G3P_HUMAN antibody
- G3PD antibody
- G3PDH antibody
- GAPD antibody
- GAPDH antibody
- Glyceraldehyde 3 phosphate dehydrogenase antibody
- Glyceraldehyde-3-phosphate dehydrogenase antibody
- HEL-S-162eP antibody
- KNC-NDS6 antibody
- MGC102544 antibody
- MGC102546 antibody
- MGC103190 antibody
- MGC103191 antibody
- MGC105239 antibody
- MGC127711 antibody
- MGC88685 antibody
- OCAS, p38 component antibody
- OCT1 coactivator in S phase, 38-KD component antibody
- peptidyl cysteine S nitrosylase GAPDH antibody
- Peptidyl-cysteine S-nitrosylase GAPDH antibody
- wu:fb33a10 antibody
see all

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

All lanes : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 2.5 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) Nuclear
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : A431 (Human epidermoid carcinoma cell line) cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate
Lane 5 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor anti-mouse at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 40.2 kDa
Observed band size: 37 kDa
why is the actual band size different from the predicted?

Fluorescence detection of secondary antibody.
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

ab8245 staining GAPDH in SV40LT-SMC cells.

The cells were fixed with 4% formaldehyde (10 minutes), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 5μg/ml and ab202272 at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150117) (shown in green). Nuclear DNA was labeled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)This image is courtesy of an anonymous Abreview

All lanes : Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

Lane 1 : Mouse hippocampus whole cell lysate
Lane 2 : Rat hippocampus whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated Rabbit anti-mouse at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40.2 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Exposure time: 10 seconds
See Abreview
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

ab8245 staining GAPDH in NIH/3T3 (Mouse embryo fibroblast cell line) cells.

The cells were fixed with 4% formaldehyde (10 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 1 μg/ml and ab202272 at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150117) (shown in green). Nuclear DNA was labeled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

ab8245 staining GAPDH in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 5 μg/ml and ab6046 at 1 µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150117) at 2 μg/ml (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)This image is courtesy of an anonymous Abreview

All lanes : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/20000 dilution

Lane 1 : Rabbit aorta, whole tissue lysate at 10 µg
Lane 2 : Blank
Lane 3 : Mouse aorta, whole tissue lysate at 5 µg
Lane 4 : Mouse aorta, whole tissue lysate at 7.5 µg
Lane 5 : Mouse aorta, whole tissue lysate at 10 µg
Lane 6 : Mouse aorta, whole tissue lysate at 12.5 µg
Lane 7 : Mouse aorta, whole tissue lysate at 15 µg

Secondary
All lanes : HRP conjugated sheep anti-mouse antibody at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40.2 kDa

Exposure time: 30 seconds
See Abreview
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 10 µg/ml + Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Predicted band size: 40.2 kDa

Immunohistochemistry protocols
Immunocytochemistry & immunofluorescence protocols
Western blot protocols

This product has been referenced in:

Xu G et al. Histone deacetylase 3 is associated with gastric cancer cell growth via the miR-454-mediated targeting of CHD5. Int J Mol Med 41:155-163 (2018). Read more (PubMed: 29115379) »
Yamada Y et al. Passenger strand of miR-145-3p acts as a tumor-suppressor by targeting MYO1B in head and neck squamous cell carcinoma. Int J Oncol 52:166-178 (2018). Read more (PubMed: 29115582) »

See all 1247 Publications for this product

Publishing research using ab8245? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 88 Abreviews

Application

Western blot

Sample

Human Cell lysate - whole cell (H1299 cells)

Gel Running Conditions

Reduced Denaturing (12)

Loading amount

25 µg

Specification

H1299 cells

Blocking step

Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 21 2018

Application

Western blot

Sample

Mouse Tissue lysate - whole (Brain, heart, liver)

Gel Running Conditions

Reduced Denaturing (12% gel)

Loading amount

25 µg

Specification

Brain, heart, liver

Blocking step

Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 21 2018

Abcam guarantees this product to work in the species/application used in this Abreview.

Application

Western blot

Sample

Human Cell lysate - other (A549)

Gel Running Conditions

Reduced Denaturing (10%)

Loading amount

50 µg

Specification

A549

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted May 22 2018

Application

Western blot

Sample

Rat Tissue lysate - whole (rat hippocampus tissue)

Gel Running Conditions

Non-reduced Denaturing

Loading amount

50 µg

Treatment

RIPA buffer

Specification

rat hippocampus tissue

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 14 2017

Application

Western blot

Sample

Bos taurus Tissue lysate - whole (Lane 1Bovine Liver and Lane 2 Bovine mammary gland)

Gel Running Conditions

Reduced Denaturing (4-15% Gradient)

Loading amount

20 µg

Specification

Lane 1Bovine Liver and Lane 2 Bovine mammary gland

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Julie Kim

Verified customer

Submitted May 12 2017

Abcam guarantees this product to work in the species/application used in this Abreview.

Application

Western blot

Sample

Mouse Tissue lysate - whole (Mouse whole brain)

Gel Running Conditions

Reduced Denaturing

Loading amount

20 µg

Specification

Mouse whole brain

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 04 2017

Abcam guarantees this product to work in the species/application used in this Abreview.

Application

Western blot

Sample

Rat Tissue lysate - whole (Liver)

Gel Running Conditions

Reduced Denaturing (12%)

Loading amount

50 µg

Specification

Liver

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 24 2017

Abcam has not validated the combination of species/application used in this Abreview.

Application

Western blot

Sample

Caenorhabditis elegans Cell lysate - whole cell (whole lysates)

Gel Running Conditions

Non-reduced Denaturing

Loading amount

50 µg

Specification

whole lysates

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Joey Wu

Verified customer

Submitted Jan 25 2017

Application

Western blot

Sample

Ferret Tissue lysate - whole (Ferret whole brain tissue)

Gel Running Conditions

Reduced Denaturing (3-8% Tris-acetate gel, NuPage)

Loading amount

40 µg

Specification

Ferret whole brain tissue

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Francis Djankpa

Verified customer

Submitted Jul 11 2016

Abcam guarantees this product to work in the species/application used in this Abreview.

Application

Western blot

Sample

Human Cell lysate - whole cell (bladder)

Gel Running Conditions

Reduced Denaturing (gel 10%)

Loading amount

30 µg

Treatment

lentivirus for gene knock-out

Specification

bladder

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Mr. Jinwoo Ahn

Verified customer

Submitted May 20 2016

1-10 of 88 Abreviews

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Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

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Western blot abreview for Anti-GAPDH antibody [6C5] - Loading Control

Western blot abreview for Anti-GAPDH antibody [6C5] - Loading Control

Western blot abreview for Anti-GAPDH antibody [6C5]

Western blot abreview for Anti-GAPDH antibody [6C5]

Western blot abreview for Anti-GAPDH antibody [6C5]

Western blot abreview for Anti-GAPDH antibody [6C5]

Western blot abreview for Anti-GAPDH antibody [6C5]

Western blot abreview for Anti-GAPDH antibody [6C5]

Western blot abreview for Anti-GAPDH antibody [6C5]

Western blot abreview for Anti-GAPDH antibody [6C5]