GAPHD Polyclonal Antibody

  • Cat.No.:E-AB-40337

  • Host: Rabbit
  • Reactivity: H,M,R
  • Applications: WB,IHC

To Purchase E-AB-40337

Size:
  • 30μL
  • 60μL
  • 120μL
  • 200μL
Price: $69
Qty:

Test Application

  • Verified Samples

    Reactivity Application
    Human WB
    (A431,HepG2,Hela,HT29,A549,Jurkat,MCF-7,)

    Western Blot analysis of A431 cells and Hela cells using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of A431 cells and Hela cells using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    IHC
    (bladder cancer,kidney,)

    Immunohistochemistry of paraffin-embedded Human bladder cancer using GAPDH Polyclonal Antibody at dilution of 1:400.

    Immunohistochemistry of paraffin-embedded Human kidney using GAPDH Polyclonal Antibody at dilution of 1:400.

    Rat WB
    (heart,brain,)

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    IHC
    (colon,)

    Immunohistochemistry of paraffin-embedded Rat colon using GAPDH Polyclonal Antibody at dilution of 1:400.

    Mouse WB
    (brain,heart,)

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    Western Blot analysis of HepG2 cells, HT29 cells, A549 cells, Jurkat cells, MCF-7 cells, Rat heart, Mouse brain, Mouse heart and Rat brain using GAPHD Polyclonal Antibody at dilution of 1:5000.

    IHC
    (colon,)

    Immunohistochemistry of paraffin-embedded Mouse colon using GAPDH Polyclonal Antibody at dilution of 1:400.

  • Dilution

    WB 1:3000-1:6000 IHC 1:300-1:500

  • Related Reagents (view more)

    Application Products
    WB Western Blot Detection Kit(E-IR-R304)
    IHC 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System (with DAB solution)(E-IR-R217)
  • Western Blot Operation Guide ( Download)

    In order to facilitate the operation and ensure the accuracy of WB results, the Western Blot Detection kit (Cat# E-IR-R304) is now available, containing the reagents which are needed from sample preparation to result detection. Please order the appropriate kit according to your specific needs.

    Separating gel12%
    Transfer Membrane150mA,1.5h
    Blocking2h
    Primary Antibody1:5000
    Secondary Antibody1:5000
    Click Here for More Details .. More ↓

Preparation of protein samples

1.Protein extraction

1)For tissue sample
a. Take the samples, wash the tissue thoroughly with pre-cooled PBS (0.01 M, pH=7.4)(Cat# E-BC-R187) to remove the surface blood and internal debris.
b. Weigh and smash the tissue, add an appropriate ratio of RIPA Lysis Buffer (Cat# E-BC-R327)(add 10 μL PMSF and 10 μL Na3VO4 to each 1 mL RIPA Lysis) and homogenizely lyse the tissue.
It is recommended to homogenize according to the ratio of tissue weight: RIPA volume = 3:10. For example, add 1 mL RIPA Lysis Buffer to 0.3 g tissue sample, the specific volume can be adjusted according to experimental requirements.
c. Shake and lyse on the ice for 30 min after homogenization. And then sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lysis and reduce the viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2)For cell sample
a. Collect the cells, wash them thoroughly with pre-cooled PBS (0.01 M, pH=7.4) to remove the medium off (it is generally recommended to wash 3 times).
b. Add an appropriate ratio of RIPA Lysate Buffer (10 μL PMSF and 10 μL Na3VO4 in each 1 mL RIPA Lysis) and lyse on the ice for 30 min.
It is recommended to add 0.1 mL of RIPA Lysis Buffer to each well of a 6-well plates (the protein content in different cells may vary, and the volume of the lysate added can be appropriately adjusted).
c. Sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lyse and reduce viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2.Measurement of protein concentration
By the BCA method (see the Total Protein Colorimetric Assay Kit (Cat# E-BC-K318) instructions).

3.Boiling the samples
Adjust the protein concentration with PBS Buffer. Add 5 × SDS Loading Buffer (Cat# E-BC-R288) with the ratio of the protein sample: 5 × SDS Loading Buffer = 4:1 and boil the mixture for 10 min. Centrifuge at 12,000 rpm for 2 min and collect the supernatant. The denatured protein can be employed to Western Blot experiments or stored at -20℃ or -80℃.

Note: It is recommended that the total protein loading amount of test sample is about 50 μg in each well. Try to make the loading volume of each sample close to 10 μL.

Electrophoresis

1.According to the molecular weight of the target protein, prepare 12% separation gel. Add the test sample to each well, and add 5 μL of Pre-stained Protein Marker (Cat# E-BC-R273)to a reserved well in order to verify the target molecular weight and the extent of membrane transfer. Add Electrophoresis Buffer ( Cat# E-BC-R331) and start electrophoresis.

2.Electrophoresis at 80v when the samples are in stacking gel, then convert to 120v when the blue flow into the separating gel. Electrophoresis time is about 2-3 h till bromophenol blue reaches the bottom of the gel.

Transfer Membrane (Wet transfer)

1.Choose the PVDF Membrane (Cat# E-BC-R266) with a pore size of 0.45 μm according to the molecular weight of the target protein. Soak the PVDF Membrane in methanol for 1 min to activate it, and then soak the PVDF Membrane in the Transmembrane Buffer (Cat# E-BC-R333), the filter paper and fiber mat must be soaked in the Transmembrane Buffer for use too.

2.Place the following materials in the order of the black plate (negative electrode) - fiber mat - filter paper - gel - PVDF Membrane - filter paper - fiber mat - white plate (positive electrode) are placed in order, discharge bubbles, clamp and place in the wet transfer tank. The recommended transmembrane conditions are 150mA,1.5h. Make sure that the transmembrane process is carried out at low temperatures.
Note: This is for wet transfer. If other transmembrane methods are used, please adjust according to the specific conditions.

3.After the transmembrane, take out the PVDF Membrane carefully and wash with TBST Buffer for 1 min.

Incubation of antibodies

1.Soak the PVDF Membrane with TBST Buffer (Cat# E-BC-R335) containing 5% Skim Milk Powder as blocking buffer and block the membrane at room temperature for 2h.

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the GAPDH Antibody at 1:5000, soak the PVDF Membrane in the primary antibody working solution, incubate overnight at 4 ℃, and gently shake.

3.Wash the PVDF Membrane with TBST Buffer for 3 times, 15 min / time..

4.According to the recommended secondary antibody dilution ratio, use a TBST Buffer solution containing 2% Skim Milk Powder to dilute Goat Anti-Rabbit IgG (H+L) (peroxidase/HRP conjugated) (Cat# E-AB-1003) at 1:5000. Incubate at room temperature for 1 h on a shaker.

5.Wash the PVDF Membrane with TBST Buffer for 3 times, 15 min / time..

Detection

1.Mix A and B in the Excellent Chemiluminescent Substrate Detection kit (Cat# E-BC-R347) at the ratio of 1:1 as working solution.

2.Take out the PVDF Membrane from TBST Buffer and absorb the liquid with the filter paper. Pave the PVDF Membrane on the detection machine, add ECL working solution continuously on the PVDF Membrane, discharge the bubble and detect the result.

3.Adjust the contrast and the exposure time to get the best image.

Appendix

Product Details

Clonality Polyclonal
Isotype IgG
Concentration 0.66mg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer PBS with 0.05% Proclin300, 50% glycerol, pH7.3.
Purification Method Antigen Affinity Purification
Research Areas Neuroscience, Isotype/Loading Controls, Signal Transduction, Cancer, Metabolism
Conjugation Unconjugated

Immunogen Details

Immunogen Recombinant Zebrafish Glyceraldehyde-3-phosphate dehydrogenase protein(PKSH500005)
Abbre GAPDH
Synonyms aging associated gene 9 protein, BARS-38, cb609, G3PDH, GAPD, KNC-NDS6, OCAS, p38 component
Swissprot Q5XJ10
Calculated MW 35 kDa
Observed MW 35 kDa
Cellular Localization Cytoplasm, Cytoskeleton, Nucleus

Background

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the phosphorylation of glyceraldehyde-3-phosphate during glycolysis. GAPDH participates in nuclear events including transcription, binding RNA, RNA transportation, DNA replication, DNA repair and apoptosis. Being stably and constitutively expressed at high levels in most tissues and cells, GAPDH is considered a housekeeping protein. It was widely used as a control for RT-PCR and also loading control in electrophoresis and Western blotting. GAPDH is normally expressed in cellular cytoplasm or membrane, but can occasionally translocate to the nucleus post modification such as S-nitrosylation.

Citations

From now on, if you have published a paper by using any of our products since 1/1/2019, fill out the “Elabscience Publication Reward Application Form”carefully and send it to orders@elabscience.com, we will get back to you with the reward after we confirm it ASAP!

View more details about our Publication Reward >>

  1. Aging (Albany NY) (2020) IF: 4.831
    K63 ubiquitin chains target NLRP3 inflammasome for autophagic degradation in ox-LDL-stimulated THP-1 macrophages

    DOI: 10.18632/aging.102710

    PMID: 32003754

    Sample: Cell culture supernatant
  2. Colloids and Surfaces B: Biointerfaces (2020) IF: 4.389
    Biological effects on tooth root surface topographies induced by various mechanical treatments

    DOI: 10.1016/j.colsurfb.2019.110748

    PMID: 31884082

    Sample: Gingival Crevicular Fluid
  3. Science of The Total Environment (2019) IF: 4.61
    Accumulation and toxicity of thiamethoxam and its metabolite clothianidin to the gonads of Eremias argus

    DOI: 10.1016/j.scitotenv.2019.02.419

    PMID: 30833257

    Sample: Plasma
  4. International Immunopharmacology (2020) IF: 3.943
    FCN-A mediates the inflammatory response and the macrophage polarization in Aspergillus fumigatus keratitis of mice by activating the MAPK signaling pathway

    DOI: 10.1016/j.intimp.2020.106473

    PMID: 32272397

    Sample: Cell lysate,Tissue homogenate
  5. Cancer medicine (2018) IF: 3.491
    ATPR- induced G 0 /G 1 phase arrest in gastric cancer cells by regulating the inding of 14- 3- 3ε and filamin A

    DOI: 10.1002/cam4.1583

    PMID: 29862660

    Sample: Cell lysate
  6. Cancer Management and Research (2020) IF: 2.886
    Different Proteins Regulated Apoptosis, Proliferation and Metastasis of Lung Adenocarcinoma After Radiotherapy at Different Time

    DOI: 10.2147/CMAR.S219967

    PMID: 32308480

    Sample: Cell lysate
  7. Biochemical and Biophysical Research Communications (2020) IF: 2.985
    Lpl-C310R mutation is associated with impaired glucose tolerance and endoplasmic reticulum stress in skeletal muscle

    DOI: 10.1016/j.bbrc.2020.06.055

    PMID: 99999999

    Sample: Plasma
  8. Neuropsychiatric disease and treatment (2019) IF: 2.157
    Microglial activation and neurobiological alterations in experimental autoimmune prostatitis-induced depressive-like behavior in mice

    DOI: 10.2147/NDT.S211288

    PMID: 31496706

    Sample: Serum,Tissue homogenate
  9. Biological trace element research (2018) IF: 2.361
    Ethanol via Regulation of NF-κB/p53 Signaling Pathway Increases Manganese-Induced Inflammation and Apoptosis in Hypothalamus of Rats

    DOI: 10.1007/s12011-018-1535-3

    PMID: 30284675

    Sample: Tissue homogenate
  10. Food Science & Nutrition (2020) IF: 1.797
    Ginsenoside Rg3 inhibits the biological activity of SGC‐7901

    DOI: 10.1002/fsn3.1707

    PMID: 99999999

    Sample: Cell lysate
  11. Archives of Oral Biology (2019) IF: 1.931
    Doxycycline inhibits NAcht Leucine-rich repeat Protein 3 inflammasome activation and interleukin-1β production induced by Porphyromonas gingivalis-lipopolysaccharide and adenosine triphosphate in human gingival fibroblasts

    DOI: 10.1016/j.archoralbio.2019.104514

    PMID: 31394382

    Sample: Cell culture supernatant
  12. Fish physiology and biochemistry (2019) IF: 1.735
    Physiological and metabolic responses of juvenile Lophiosilurus alexandri catfish to air exposure

    DOI: 10.1007/s10695-018-0576-z

    PMID: 30368686

    Sample: Plasma
  13. Andrologia (2019) IF: 1.84
    Effects of Hydrogen Sulphide Donor, Sodium Hydrosulphide Treatment on the Erectile Dysfunction in L-NAME-induced Hypertensive Rats

    DOI: 10.1111/and.13240

    PMID: 30706510

    Sample: Tissue homogenate
  14. Preprint (2019)
    The neuroprotective effects of xenon on neonatal rats with white matter damage by regulating expression of microRNA-210 and HIF-1α

    DOI: 10.21203/rs.2.10732/v1

    PMID: 99999999

    Sample: Tissue homogenate
  15. International journal of ophthalmology (2020)
    Regulation of LOX-1 on adhesion molecules and neutrophil infiltration in mouse Aspergillus fumigatus keratitis

    DOI: 10.18240/ijo.2020.06.03

    PMID: 32566496

    Sample: Tissue homogenate,Tissue Slice
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Reviews/Q&A

  • Show all
  • Reviews
  • Q&A

Verified Customer

Q********pSubmitted [ Jan 10 2020 ]

  • Application:WB
  • Species:Human
  • Loading amount:10μg
  • Sample source:HepG2 Jurkat
  • Gel Running Conditions:Reduced, Denaturing, Other details:12%
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1hours 30minutes

  • Primary antibody:

    Dilution:1:5000

    Time: 17hours

  • Temperature:4℃

    Diluent:5% Milk

  • Secondary antibody:Use Elabscience secondary antibody
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Western Blot analysis of HepG2 and Jurkat using GAPDH Polycloanl Antibody at dilution of 1:5000.This product works great.
Read More
Read Less

Verified Customer

D*****uSubmitted [ Dec 12 2019 ]

  • Application:WB
  • Species:Human
  • Loading amount:10μg
  • Sample source:A431 Hela
  • Gel Running Conditions:Reduced, Denaturing, Other details:12%
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1hours 30minutes

  • Primary antibody:

    Dilution:1:2000

    Time: 18hours

  • Temperature:4℃

    Diluent:5% Milk

  • Secondary antibody:Use Elabscience secondary antibody
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Western Blot analysis of A431 and Hela cells using GAPDH Polyclonal Antibody at dilution of 1:2000.
Read More
Read Less

Verified Customer

P***rSubmitted [ Nov 21 2019 ]

  • Application:WB
  • Species:Human
  • Loading amount:50μg
  • Sample source:A549、Jurkat
  • Gel Running Conditions:Reduced, Denaturing, Other details:12
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1.5hours

  • Primary antibody:

    Dilution:1:5000

    Time: overnhours

  • Temperature:4℃

    Diluent:5% skim milk

  • Secondary antibody:Use Elabscience secondary antibody E-AB-1003
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Lane 1:A549 Lane 2:Jurkat This product works great.The bands are about 35 kDa.
Read More
Read Less
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