Name: Anti-GAPDH antibody [6C5] - Loading Control
SKU: ab8245
Rating: 5 (100 reviews)

Anti-GAPDH antibody 6C5 is a mouse monoclonal antibody that is used in GAPDH Western blot, ICC/IF. Suitable for Human, Mouse, Rat samples.

- Preliminary data suggests ab8245 recognises GAPDH monomer and dimer forms, but not tetrameric forms
- Loading control antibody
- Cited in over 5,100 publications

Images

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245), expandable thumbnail

Publications

Key facts

Isotype: IgG1
Host species: Mouse
Storage buffer: pH: 7.4
Preservative: 0.09% Sodium azide
Constituents: PBS
Form: Liquid
Clonality: Monoclonal

Immunogen

Native Full Length Protein corresponding to Rabbit GAPDH. Database link P46406

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	WB	ICC/IF
Human	Tested	Tested
Mouse	Expected	Tested
Rat	Expected	Tested
Baboon	Not recommended	Not recommended
Cat	Not recommended	Not recommended
Chicken	Not recommended	Not recommended
Cow	Not recommended	Not recommended
Dog	Not recommended	Not recommended
Fish	Not recommended	Not recommended
Goat	Not recommended	Not recommended
Guinea pig	Not recommended	Not recommended
Hamster	Not recommended	Not recommended
Horse	Not recommended	Not recommended
Monkey	Not recommended	Not recommended
Pig	Not recommended	Not recommended
Saccharomyces cerevisiae	Not recommended	Not recommended
Xenopus laevis	Not recommended	Not recommended
Zebrafish	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500.00000 - 1/10000.00000	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Saccharomyces cerevisiae, Cow, Goat, Horse, Chicken, Guinea pig, Hamster, Cat, Dog, Pig, Xenopus laevis, Fish, Monkey, Zebrafish, Baboon	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1.00000-5.00000 µg/mL	Notes -
Species Rat	Dilution info 1.00000-5.00000 µg/mL	Notes -
Species Human	Dilution info 1.00000-5.00000 µg/mL	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Saccharomyces cerevisiae, Cow, Goat, Horse, Chicken, Guinea pig, Hamster, Cat, Dog, Pig, Xenopus laevis, Fish, Monkey, Zebrafish, Baboon	Dilution info -	Notes -

Associated Products

Select an associated product type

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Target data

See full target information GAPDH

Function

Has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively (PubMed:11724794, PubMed:3170585). 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 (PubMed:11724794, PubMed:3170585). Modulates the organization and assembly of the cytoskeleton (By similarity). Facilitates the CHP1-dependent microtubule and membrane associations through its ability to stimulate the binding of CHP1 to microtubules (By similarity). Component of the GAIT (gamma interferon-activated inhibitor of translation) complex which mediates interferon-gamma-induced transcript-selective translation inhibition in inflammation processes (PubMed:23071094). Upon interferon-gamma treatment assembles into the GAIT complex which binds to stem loop-containing GAIT elements in the 3'-UTR of diverse inflammatory mRNAs (such as ceruplasmin) and suppresses their translation (PubMed:23071094). Also plays a role in innate immunity by promoting TNF-induced NF-kappa-B activation and type I interferon production, via interaction with TRAF2 and TRAF3, respectively (PubMed:23332158, PubMed:27387501). Participates in nuclear events including transcription, RNA transport, DNA replication and apoptosis (By similarity). 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).

Alternative names

GAPD, CDABP0047, OK/SW-cl.12, GAPDH, Glyceraldehyde-3-phosphate dehydrogenase, Peptidyl-cysteine S-nitrosylase GAPDH

Key facts

Isotype: IgG1
Host species: Mouse
Storage buffer: pH: 7.4
Preservative: 0.09% Sodium azide
Constituents: PBS
Form: Liquid
Clonality: Monoclonal
Immunogen: Native Full Length Protein corresponding to Rabbit GAPDH. Database link P46406
Clone number: 6C5
Purification technique: Affinity purification Protein A
Specificity: This GAPDH antibody can be used as a loading control antibody. 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 GAPDH antibody- loading control ab8245 recognizes the monomer (36 kDa) and also the dimer forms of GAPDH, but not the tetrameric form of the protein.
Concentration
Purification notes: Chromatography on protein A Sepharose

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Anti-GAPDH antibody [6C5] - Loading Control (ab8245) is a mouse monoclonal antibody and is validated for use in ICC/IF and WB.

Anti-GAPDH antibody [6C5] - Loading Control (ab8245) was first used in a scientific publication in 2003 and has been cited over 5105 times in peer reviewed journals. It's performance in Western Blot in human, mouse and rat samples is trusted by the scientific community.

Abcam's high quality validation processes ensure Anti-GAPDH antibody [6C5] - Loading Control (ab8245) has high sensitivity and specificity.

Anti-GAPDH antibody [6C5] - Loading Control (ab8245) has 100 independent reviews from customers.

GAPDH antibodies are often used as loading controls in Western Blot. Anti-GAPDH antibody [6C5] - Loading Control has been verified in Western Blot samples and detects a band at 36kDa Molecular weight.

Anti-GAPDH antibody [6C5] - Loading Control (ab8245) specifically detects GAPDH (UniProt ID: P46406; Molecular weight: 36kDa) and is sold in 100 µg selling sizes.

One of the top cited antibody in the market for GAPDH with >6500 citations and >70 five star reviews. GAPDH is a key target involved in glycolysis and cell metabolism. It plays a crucial role as a housekeeping gene, particularly in understanding GAPDH expression and its function as a metabolic enzyme. GAPDH is widely analysed in GAPDH activity assays and studies of its role in various cellular processes. Additionally, GAPDH is commonly used as a loading control in Western blot and immunocytochemistry/immunofluorescence (ICC/IF) experiments.

Abcam is leading the way to address reproducibility in scientific research with our highly validated recombinant monoclonal and recombinant multiclonal antibodies. Search & select one of Abcam's thousands of recombinant alternatives to eliminate batch-variability and unnecessary animal use.

If you do not find a host species to meet your needs, our catalogue and custom Chimeric range provides scientists the specificity of Abcam's RabMAbs in the species backbone of your choice. Remember to also review our range of edited cell lines, proteins and biochemicals relevant to your target that may help you further your research goals.

Abcam antibodies are extensively validated in a wide range of species and applications, so please check the reagent specifications meet your scientific needs before purchasing. If you have any questions or bespoke requirements, simply visit the Contact Us page to send us an inquiry or contact our Support Team ahead of purchase.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

GAPDH also known as glyceraldehyde 3-phosphate dehydrogenase plays a mechanical role in the glycolytic pathway where it catalyzes the sixth step converting glyceraldehyde 3-phosphate into 13-bisphosphoglycerate. This enzyme has a molecular weight of about 36 kDa. GAPDH is ubiquitously expressed in many tissues and cells making it an extensively studied protein in various biological processes. Due to its consistent expression level researchers often use GAPDH as a loading control in western blot experiments to ensure equal protein loading across samples.

Biological function summary

Glyceraldehyde 3-phosphate dehydrogenase contributes not only to energy production through glycolysis but also has roles beyond metabolism. It connects to cellular functions such as apoptosis and acts as a co-factor in RNA binding. Although it is not typically part of a stable protein complex its involvement in numerous cellular functions highlights its importance in maintaining cellular homeostasis.

Pathways

Glyceraldehyde 3-phosphate dehydrogenase integrates into glycolysis the central metabolic pathway for energy production in cells. Besides glycolysis it links to the regulation of apoptosis working alongside proteins like Bcl-2 which modulate cell survival. These pathways demonstrate the protein's critical role in balancing cell energy requirements and programmed cell death.

Associated diseases and disorders

Abnormalities in GAPDH expression and function relate to neurodegenerative conditions such as Alzheimer's disease and cancer. In Alzheimer's disease GAPDH interactions with proteins like amyloid-beta and tau proteins exacerbate neuronal damage. When overexpressed or dysfunctional in cancer GAPDH supports rapid cancer cell growth and proliferation by enhancing glycolytic flux a behavior known as the Warburg effect.

Product promise

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37 product images

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab140751 overnight at 4°C. Antibody binding was detected using the Donkey Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216779 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/2000 dilution
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NF-κB p60 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)
Secondary
All lanes: Western blot - Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216779) at 1/10000 dilution
Predicted band size: 36 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
Lane 1: Wild-type HeLa cell lysate (20µg)

Lane 2: SORT1 knockout HeLa cell lysate (20µg)

Lanes 1- 2: Merged signal (red and green). Green - Anti-Sortilin/NT3 antibody [EPR15010] ab188586 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.

Anti-Sortilin/NT3 antibody [EPR15010] ab188586 Anti-Sortilin/NT3 antibody [EPR15010] was shown to specifically react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human SORT1 (Sortilin/NT3) knockout HeLa cell line ab264772 (knockout cell lysate Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) was used. Wild-type and Sortilin/NT3 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-Sortilin/NT3 antibody [EPR15010] ab188586 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4^°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (Anti-Sortilin/NT3 antibody [EPR15010] ab188586) at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) at 20 µg
Secondary
Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution
Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 92 kDa
Observed band size: 100 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
Lanes 1- 2: Merged signal (red and green). Green - Anti-Sortilin/NT3 antibody ab16640 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
Anti-Sortilin/NT3 antibody ab16640 was shown to react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human SORT1 (Sortilin/NT3) knockout HeLa cell line ab264772 (knockout cell lysate Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) was used. Wild-type HeLa and SORT1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-Sortilin/NT3 antibody ab16640 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 μg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Sortilin/NT3 antibody (Anti-Sortilin/NT3 antibody ab16640) at 1 µg/mL
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) at 20 µg
Secondary
Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution
Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 92 kDa
Observed band size: 100 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
False colour image of Western blot: Anti-SIRPA antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to SIRPA. A band was observed at 100-140 kDa (mouse SIRPA, isoform 1), in wild-type RAW 264.7 cell lysates (band observed at 70-100 kDa in THP-1 is Human SIRPA) with no signal observed at this size in SIRPA knockout cell line Mouse SIRPA knockout RAW 264.7 cell line ab281618 (knockout cell lysate Mouse SIRPA knockout RAW 264.7 cell lysate ab282969). To generate this image, wild-type and SIRPA knockout RAW 264.7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/20000 dilution
Lane 1: Wild-type RAW 264.7 cell lysate at 20 µg
Lane 2: RAW 264.7 cell lysate at 20 µg
Lane 2: Western blot - Mouse SIRPA knockout RAW 264.7 cell line (Mouse SIRPA knockout RAW 264.7 cell line ab281618)
Lane 3: THP-1 cell lysate at 20 µg
Lane 4: MCF7 cell lysate at 20 µg
Predicted band size: 55 kDa
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
GAPDH immunofluorescence staining of HeLa cells using mouse anti-GAPDH antibody

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 Anti-beta Tubulin antibody - Loading Control 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 (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 (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.
This image is courtesy of an anonymous customer review
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
Lane 1: Mouse hippocampus whole cell lysate at 20 µg
Lane 2: Rat hippocampus whole cell lysate at 20 µg
Secondary
All lanes: HRP-conjugated Rabbit anti-mouse at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 36 kDa
Observed band size: 36 kDa
Exposure time: 10s
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with unpurified Anti-BDNF antibody [EPR1292] ab108319 (1/1000) overnight at 4°C. ab8245 (mouse anti-GAPDH; 0.05 ug/mL) was included as a loading control. Antibody binding was detected using goat anti-rabbit IgG IR-680 (green) and goat anti-mouse IgG IR800 (red) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx
All lanes: Western blot - Anti-BDNF antibody [EPR1292] (Anti-BDNF antibody [EPR1292] ab108319) at 1/1000 dilution
Lane 1: Human hippocampus lysate at 20 µg
Lane 2: Rat hippocampus lysate at 20 µg
Lane 3: Mouse hippocampus lysate at 20 µg
Secondary
All lanes: Gt anti Rb IR680 at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 27 kDa
Observed band size: 15 kDa, 28 kDa, 35 kDa, 45 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with Anti-Brd4 antibody [EPR5150(2)] ab128874 overnight at 4°C. Antibody binding was detected using the Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773. Loading control to GAPDH ab8245 antibody binding was detected using the Goat anti-Mouse IgG H&L (IRDye^® 680RD) (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
Merged signal (red and green). Green Anti-Brd4 antibody [EPR5150(2)] Anti-Brd4 antibody [EPR5150(2)] ab128874 observed at 150 kDa using Goat anti-Rabbit IgG H&L (IRDye^® 800CW)-Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 as secondary antibody. Red - Anti-GAPDH antibody loading control, ab8245, observed at 37 kDa,
Lane 1: Western blot - Anti-Brd4 antibody [EPR5150(2)] (Anti-Brd4 antibody [EPR5150(2)] ab128874)
Lanes 1 - 3: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/10000 dilution
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Brd4 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Secondary
All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution
Predicted band size: 152 kDa, 36 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
Lane 1: Wild-type HCT116 cell lysate 20 ug
Lane 2: CTNNB1 knockout HCT116 cell lysate 20 ug
Lanes 1 - 2: Merged signal (red and green). Green - Anti-beta Catenin antibody [IGX4794R-3] ab223075 observed at 95 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
Anti-beta Catenin antibody [IGX4794R-3] ab223075 was shown to react with Anti-beta Catenin in wild-type HCT 116 cells in western blot with loss of signal observed in CTNNB1 knockout cell line Human CTNNB1 (beta Catenin II) knockout HCT116 cell line ab273712 (CTNNB1 knockout cell lysate Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247). HCT 116 wild-type and CTNNB1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluoroscent western blot (TBS-based) blocking solution 50% (v/v) in TBS-T (0.1% Tween^®) before incubation with Anti-beta Catenin antibody [IGX4794R-3] ab223075 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4^°C at 1 ug/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-beta Catenin antibody [IGX4794R-3] (Anti-beta Catenin antibody [IGX4794R-3] ab223075) at 1 µg/mL
Lane 1: Wild-type HCT116 cell lysate at 20 µg
Lane 2: Western blot - Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate (Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247)
Lane 2: Western blot - Human CTNNB1 (beta Catenin II) knockout HCT116 cell line (Human CTNNB1 (beta Catenin II) knockout HCT116 cell line ab273712)
Lane 2: CTNNB1 knockout HCT116 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 85 kDa
Observed band size: 95 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 10 µg/mL
All lanes: Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg
Predicted band size: 36 kDa
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
GAPDH immunofluorescence staining of 3T3 cells using mouse anti-GAPDH antibody

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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab202272 at 1/250 dilution 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 (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)
Lane 1: Wild-type HCT 116 cell lysate 20 μg
Lane 2: CTNNB1 knockout HCT 116 cell lysate 20 μg
False colour image of Western blot: Anti-beta Catenin antibody [IGX4794R-3] staining at 1 μg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-beta Catenin antibody [IGX4794R-3] ab223075 was shown to bind specifically to beta Catenin. A band was observed at 95 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CTNNB1 knockout cell line Human CTNNB1 (beta Catenin II) knockout HCT116 cell line ab273712 (knockout cell lysate Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247). The band observed in the knockout lysate lane below 95 kDa is likely to represent a truncated form of beta Catenin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CTNNB1 knockout HCT 116 cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-beta Catenin antibody [IGX4794R-3] (Anti-beta Catenin antibody [IGX4794R-3] ab223075) at 1 µg/mL
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: Western blot - Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate (Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247)
Lane 2: Western blot - Human CTNNB1 (beta Catenin II) knockout HCT116 cell line (Human CTNNB1 (beta Catenin II) knockout HCT116 cell line ab273712)
Lane 2: CTNNB1 knockout HCT 116 cell lysate at 20 µg
Secondary
Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution
Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 85 kDa
Observed band size: 95 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
Fluorescence detection of secondary antibody.
All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 2.5 µg/mL
Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) Nuclear at 20 µg
Lane 2: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 3: A431 (Human epidermoid carcinoma cell line) cell lysate at 20 µg
Lane 4: Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate at 20 µg
Lane 5: HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate at 20 µg
Secondary
All lanes: Alexa Fluor anti-mouse at 1/5000 dilution
Performed under reducing conditions.
Predicted band size: 36 kDa
Observed band size: 37 kDa
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
GAPDH immunofluorescence staining of SC40LT-SMC cells using mouse anti-GAPDH antibody

ab8245 staining GAPDH in SV40LT-SMC (Rat SV40-transfected aorta smooth cell line) 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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab202272 at 1/250 dilution 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 (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)
Anti-EZH2 antibody [EPR24902-112] ab287893 was shown to specifically react with EZH2 (pT345) in HEK-293. The signal was significantly reduced after blocking with 1µg/ml histone-lysine N-methyltransferase EZH2 peptide (pT345). Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% BSA in TBS-0.1 % Tween® 20 (TBS-T). Anti-EZH2 antibody [EPR24902-112] ab287893 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilutions, respectively. Blots were developed with Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-EZH2 antibody [EPR24902-112] (Anti-EZH2 antibody [EPR24902-112] ab287893) at 1/1000 dilution
Lane 1: HEK-293 cell lysate at 20 µg
Lane 2: HEK-293 cell lysate with histone-lysine N-methyltransferase EZH2 peptide (unmodified) at 1µg/ml at 20 µg
Lane 3: HEK-293 cell lysate with histone-lysine N-methyltransferase EZH2 peptide (pT345) at 1µg/ml at 20 µg
Secondary
Lanes 1 - 3: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 3: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 98 kDa, 37 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-PICK1 antibody [EPR25155-48] (Anti-PICK1 antibody [EPR25155-48] ab290727) at 1/1000 dilution
Lane 1: Wild-type U-87 MG at 20 µg
Lane 3: T-47D at 20 µg
Lane 4: MCF7 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 47 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-C1s antibody [EPR9066(B)] (Anti-C1s antibody [EPR9066(B)] ab134943) at 1/1000 dilution
Lane 1: Wild-type A549 at 20 µg
Lane 2: C1S knockout A549 at 20 µg
Lane 3: HEK-293 at 20 µg
Lane 4: Human Liver at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 77 kDa
Observed band size: 77 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (Anti-SP1 antibody [EPR22648-50] - ChIP Grade ab231778) at 1/1000 dilution
Lane 1: Wild-type MCF7 at 20 µg
Lane 2: SP1 knockout MCF7 at 20 µg
Lane 3: HeLa at 20 µg
Lane 4: Ramos at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 90 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-KIF5A antibody (Anti-KIF5A antibody ab5628) at 1/1000 dilution
Lane 1: Wild-type U-87 MG ab278079 at 20 µg
Lane 2: KIF5A knockout U-87 MG ab306717 at 20 µg
Lane 3: SK-N-FI at 20 µg
Lane 4: HEK-293T at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 117 kDa
Observed band size: 135 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Anti-CHCHD10 antibody produced in rabbit at 1/1000 dilution
Lane 1: Wild-type A549 ab288558 at 20 µg
Lane 2: CHCHD10 knockout A549 at 20 µg
Lane 3: LNCaP at 20 µg
Lane 4: U-2 OS at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 14 kDa
Observed band size: 18 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-TRIM8 antibody [EPR27965-6] (Anti-TRIM8 antibody [EPR27965-6] ab316149) at 1/1000 dilution
Lane 1: Wild-type MCF7 at 20 µg
Lane 2: TRIM8 knockout MCF7 at 20 µg
Lane 3: OVCAR-3 at 20 µg
Lane 4: HCT 116 at 20 µg
Lane 5: HepG2 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 65 kDa
Observed band size: 65 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-Presenilin 2/AD5 antibody [EP1515Y] (Anti-Presenilin 2/AD5 antibody [EP1515Y] ab51249) at 1/20000 dilution
Lane 1: Wild-type U-87 MG ab278079 at 20 µg
Lane 2: PSEN2 knockout U-87 MG at 20 µg
Lane 3: A549 at 20 µg
Lane 4: MOLT-4 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 50 kDa
Observed band size: 18 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-ACSS2 antibody [EPR27496-4] (Anti-ACSS2 antibody [EPR27496-4] ab314490) at 1/1000 dilution
Lane 1: Wild-type U-87 MG ab278079 at 20 µg
Lane 2: ACSS2 knockout U-87 MG at 20 µg
Lane 3: Wild-type HAP1 at 20 µg
Lane 4: ACSS2 knockout HAP1 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 79 kDa
Observed band size: 75 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-TBC1D15 antibody - N-terminal (Anti-TBC1D15 antibody - N-terminal ab194953) at 1/1000 dilution
Lane 1: Wild-type A549 ab288558 at 20 µg
Lane 2: TBC1D15 knockout A549 at 20 µg
Lane 3: HeLa at 20 µg
Lane 4: A549 Nuclear at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 79 kDa
Observed band size: 75 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-TBC1D15 antibody (Anti-TBC1D15 antibody ab121396) at 0.1 µg/mL
Lane 1: Wild-type A549 ab288558 at 20 µg
Lane 2: TBC1D15 knockout A549 at 20 µg
Lane 3: HeLa at 20 µg
Lane 4: A549 Nuclear at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 79 kDa
Observed band size: 75 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-ACSS2 antibody [EPR8500] (Anti-ACSS2 antibody [EPR8500] ab133664) at 1/1000 dilution
Lane 1: Wild-type U-87 MG ab278079 at 20 µg
Lane 2: ACSS2 knockout U-87 MG at 20 µg
Lane 3: Wild-type HAP1 at 20 µg
Lane 4: ACSS2 knockout HAP1 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 79 kDa
Observed band size: 75 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Akt3 (E1Z3W) Rabbit mAb at 1/1000 dilution
Lane 1: Wild-type A549 ab288558 at 20 µg
Lane 2: AKT3 knockout A549 Human AKT3 knockout A549 cell line ab286451 at 20 µg
Lane 3: THP-1 at 20 µg
Lane 4: A549 Cytoplasmic at 20 µg
Lane 5: Recombinant Human AKT3 protein at 0.2 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 54 kDa
Observed band size: 60 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
GAPDH Western blot staining using mouse Anti-GAPDH antibody
Lysates at 20 µg per lane.
The samples were run on a Bis-Tris gel.
Performed under reducing conditions.
False colour image of Western blot: Anti-SUZ12 antibody ( Anti-SUZ12 antibody [EPR26230-82] ab307891) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, Anti-SUZ12 antibody [EPR26230-82] ab307891 was shown to bind specifically to SUZ12. A band was observed at 95 kDa in wild-type HAP1 cell lysates with no signal observed at this size in SUZ12 knockout cell line. To generate this image, wild-type and SUZ12 knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
Lane 1: Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 20 µg
Lane 2: SUZ12 knockout HAP1 whole cell lysate at 20 µg
Lane 3: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 4: Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Predicted band size: 83 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
GAPDH Western blot staining using mouse Anti-GAPDH antibody
Lanes 1 - 3: Western blot - Anti-KIFC1 antibody [11445] (Anti-KIFC1 antibody [11445] ab172620) at 1/50000 dilution
Lanes 1 - 4: Western blot - Anti-PERK antibody [EPR19876-294] - BSA and Azide free (Anti-PERK antibody [EPR19876-294] - BSA and Azide free ab254249) at 1/50000 dilution
Lane 1: Wild-type A549 at 20 µg
Lane 2: KIFC1 knockout A549 at 20 µg
Lane 2: KIFC1 knockout A549 at 20 g
Lane 3: U-87 MG at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 74 kDa
Observed band size: 75 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-KIF5A antibody (Anti-KIF5A antibody ab154378) at 1/1000 dilution
Lane 1: Wild-type U-87 MG ab278079 at 20 µg
Lane 2: KIF5A knockout U-87 MG ab306717 at 20 µg
Lane 3: SK-N-FI at 20 µg
Lane 4: HEK-293T at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 117 kDa
Observed band size: 135 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-MALT1/MLT antibody [EP603Y] (Anti-MALT1/MLT antibody [EP603Y] ab33921) at 1/1000 dilution
Lane 1: Wild-type HCT 116 ab288559 at 20 µg
Lane 2: MALT1 knockout HCT 116 Human MALT1 knockout HCT116 cell line ab286597 at 20 µg
Lane 3: HeLa at 20 µg
Lane 4: Ramos at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 92 kDa
Observed band size: 95 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-Furin antibody (Anti-Furin antibody ab3467) at 1/1000 dilution
Lane 1: Wild-type A549 at 20 µg
Lane 2: FURIN knockout A549 at 20 µg
Lane 3: HEK-293 at 20 µg
Lane 4: MOLT-4 at 20 µg
Lane 5: Ramos at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 87 kDa
Observed band size: 87 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-MALT1/MLT antibody [EPR24445-129] (Anti-MALT1/MLT antibody [EPR24445-129] ab283573) at 1/1000 dilution
Lane 1: Wild-type HCT 116 ab288559 at 20 µg
Lane 2: MALT1 knockout HCT 116 Human MALT1 knockout HCT116 cell line ab286597 at 20 µg
Lane 3: HeLa at 20 µg
Lane 4: Ramos at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 92 kDa
Observed band size: 95 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot - Anti-MeCP2 antibody - ChIP Grade (Anti-MeCP2 antibody - ChIP Grade ab195393) at 1/1000 dilution
Lane 1: Wild-type HCT 116 ab288559 at 20 µg
Lane 2: MECP2 knockout HCT 116 Human MECP2 knockout HCT116 cell line ab289193 at 20 µg
Lane 3: Wild-type HAP1 at 20 µg
Lane 4: MECP2 knockout HAP1 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 52 kDa
Observed band size: 52 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
All lanes: Western blot at 1/1000 dilution
Lane 1: Wild-type A549 at 20 µg
Lane 2: FURIN knockout A549 at 20 µg
Lane 3: HEK-293 at 20 µg
Lane 4: MOLT-4 at 20 µg
Lane 5: Ramos at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 87 kDa
Observed band size: 87 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
GAPDH Western blot staining using mouse Anti-GAPDH antibody
All lanes: Western blot - Anti-S6K1 antibody [E343] (Anti-S6K1 antibody [E343] ab32529) at 1/10000 dilution
Lane 1: Wild-type U-87 MG at 20 µg
Lane 2: RPS6KB1 knockout U-87 MG at 20 µg
Lane 3: MCF7 at 20 µg
Lane 4: HEK-293 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 59 kDa
Observed band size: 59 kDa
Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)
Western blot: Anti-FMRP antibody Anti-FMRP antibody ab17722 staining at 1 µg/mL, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 75 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in FMR1 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.
All lanes: Western blot - Anti-FMRP antibody (Anti-FMRP antibody ab17722) at 1 µg/mL
Lane 1: Wild-type U-87 MG at 20 µg
Lane 2: Western blot - Human FMR1 knockout U-87 MG cell line (Human FMR1 knockout U-87 MG cell line ab306664) at 20 µg
Lane 3: Wild-type A549 at 20 µg
Lane 4: Western blot - Human FMR1 knockout A549 cell line (Human FMR1 knockout A549 cell line ab288956) at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 75 kDa