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Goat Anti-Rabbit IgG (H+L)(peroxidase/HRP conjugated)

Cat.No.:E-AB-1003

Host: Goat

Applications: WB,IHC,ELISA

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Test Application

  • Verified Samples

    Reactivity Application
  • Dilution

    Western Blotting: 1:5,000-50,000 (Enhanced chemiluminescent detection ) Western Blotting: 1:1,000-5,000(DAB detection) Direct ELISA: 1:4,000-8,000(TMB detection) Immunohisto/cytochemistry: 1:400-4,000(DAB detection)

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 0% 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# ) with a pore size of 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 . 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 .

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the Antibody at , 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 .

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 . Incubate at room temperature for 1 h on a shaker.

5.Wash the PVDF Membrane with TBST Buffer for .

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
Concentration 0.5 mg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer PBS with 50% glycerol, PH7.4
Purification Method Affinity purification
Conjugation HRP

Immunogen Details

Immunogen Rabbit IgG
Synonyms Goat Anti-Rabbit IgG

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!

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  1. Cell reports (2019) IF: 8.109
    SRSF11 Loss Leads to Aging-Associated Cognitive Decline by Modulating LRP8 and ApoE

    DOI: 10.1016/j.celrep.2019.06.002

    PMID: 31269452

    Sample: Cell sample
  2. Nano Research (2016) IF: 7.354
    The Influence Of Nanotopography On Organelle Organization And Communication.

    DOI: 10.1007/s12274-016-1129-3

    Sample: Cell lysate
  3. International journal of biological macromolecules (2020) IF: 5.162
    Anti-inflammatory effect of epidermal growth factor conjugated silk fibroin immobilized polyurethane ameliorates diabetic burn wound healing

    DOI: 10.1016/j.ijbiomac.2019.09.219

    PMID: 31647938

    Sample: Serum,Tissue homogenate
  4. 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
  5. International journal of molecular sciences (2019) IF: 4.556
    Anti-Diabetic Nephropathy Activities of Polysaccharides Obtained from Termitornyces albuminosus via Regulation of NF-κB Signaling in db/db Mice

    DOI: 10.3390/ijms20205205

    PMID: 31640118

    Sample: Tissue homogenate
  6. Immunology and cell biology (2020) IF: 3.745
    Dimethyl itaconate protects against fungal keratitis by activating the Nrf2/HO-1 signaling pathway

    DOI: 10.1111/imcb.12316

    PMID: 31943336

    Sample: Cell culture supernatant,Tissue homogenate
  7. International Immunopharmacology (2019) IF: 3.118
    The role of netrin-1 in the mouse cornea during Aspergillus fumigatus infection

    DOI: 10.1016/j.intimp.2019.03.047

    PMID: 30952101

    Sample: Tissue homogenate,Cell lysate
  8. Toxicology (2019) IF: 3.265
    Ethanol increases manganese-Induced spatial learning and memory deficits via oxidative/nitrosative stress induced p53 dependent/independent hippocampal apoptosis

    DOI: 10.1016/j.tox.2019.03.001

    PMID: 30844427

    Sample: Tissue homogenate
  9. Human & Experimental Toxicology (2020) IF: 2.067
    SIRT4 prevents excitotoxicity via modulating glutamate metabolism in glioma cells

    DOI: 10.1177/0960327120907142

    PMID: 32081049

    Sample: Cell lysate
  10. Neuroscience Letters (2020) IF: 2.274
    Intrathecal administration of SRT1720 relieves bone cancer pain by inhibiting the CREB/CRTC1 signalling pathway

    DOI: 10.1016/j.neulet.2019.134623

    PMID: 31722235

    Sample: Tissue homogenate
  11. Toxicon (2020) IF: 2.201
    Botulinum toxin type A ameliorates adjuvant-arthritis pain by inhibiting microglial activation-mediated neuroinflammation and intracellular molecular signaling

    DOI: 10.1016/j.toxicon.2019.12.153

    PMID: 32250746

    Sample: Tissue homogenate
  12. Applied biochemistry and biotechnology (2020) IF: 2.277
    Studies on the Neuroprotection of Osthole on Glutamate-Induced Apoptotic Cells and an Alzheimer's Disease Mouse Model via Modulation Oxidative Stress

    DOI: 10.1007/s12010-019-03101-2

    PMID: 31407160

    Sample: Cell lysate
  13. Neuroscience letters (2019) IF: 2.159
    PI3K/Akt signaling pathway may be involved in MCP-1-induced P2X4R expression in cultured microglia and cancer-induced bone pain rats

    DOI: 10.1016/j.neulet.2019.02.024

    PMID: 30776493

    Sample: Tissue homogenate
  14. Biomedicine & Pharmacotherapy (2017) IF: 2.759
    Triptonide Inhibits The Pathological Functions Of Gastric Cancer-Associated Fibroblasts.

    DOI: 10.1016/j.biopha.2017.10.046

    PMID: 29049979

  15. Pharmacology (2020) IF: 1.625
    Gastrodin Alleviates Vascular Dementia in a 2-VO-Vascular Dementia Rat Model by Altering Amyloid and Tau Levels

    DOI: 10.1159/000504056

    PMID: 31752010

    Sample: Tissue homogenate
  16. The Journal of Spinal Cord Medicine (2019) IF: 1.816
    Assessment of the neuroprotective effects of (-)-epigallocatechin-3-gallate on spinal cord ischemia-reperfusion injury in rats

    DOI: 10.1080/10790268.2019.1691862

    PMID: 31809244

    Sample: Tissue homogenate
  17. Toxicology research (2018) IF: 1.89
    Roles of ERK1/2 and PI3K/AKT signaling pathways in mitochondria-mediated apoptosis in testes of hypothyroid rats

    DOI: 10.1039/c8tx00122g

    PMID: 30542605

    Sample: Tissue homogenate
  18. Oncology letters (2019) IF: 1.664
    Exosome-delivered TRPP2 siRNA Inhibits the Epithelial-Mesenchymal Transition of FaDu Cells

    DOI: 10.3892/ol.2018.9752

    PMID: 30675260

    Sample: FaDu cells
  19. Experimental and therapeutic medicine (2019) IF: 1.41
    Effect of PI3K/Akt/mTOR signaling pathway on JNK3 in Parkinsonian rats

    DOI: 10.3892/etm.2018.7120

    PMID: 30783448

    Sample: Tissue homogenate
  20. Molecular medicine reports (2019) IF: 1.922
    Ginsenoside Rg5 induces apoptosis in human esophageal cancer cells through the phosphoinositide-3 kinase/protein kinase B signaling pathway

    DOI: 10.3892/mmr.2019.10093

    PMID: 30942438

    Sample: Cell lysate
  21. Oncology letters (2018) IF: 1.664
    Dihydroartemisinin Triggers c-Myc Proteolysis and Inhibits Protein Kinase B/glycogen Synthase Kinase 3β Pathway in T-cell Lymphoma Cells

    DOI: 10.3892/ol.2018.9450

    PMID: 30405828

    Sample: Cells
  22. Human & Experimental Toxicology (2018) IF: 1.84
    Influence of hypothyroidism on testicular mitochondrial oxidative stress by activating the p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase signaling pathways in rats.

    DOI: 10.1177/0960327118781927

    PMID: 29896988

  23. Preprint (2020)
    hnRNPM Deficiency Deteriorates Cognitive Function by Decreasing Synaptic Proteins

    DOI: 10.21203/rs.3.rs-34523/v1

    PMID: 99999999

    Sample: Cell culture supernatant
  24. 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
  25. Carbohydr Polym (2020)
    Studies on characteristics and anti-diabetic and -nephritic effects of polysaccharides isolated from Paecilomyces hepiali fermentation mycelium in db/db mice

    DOI: 10.1016/j.carbpol.2019.115766

    PMID: 31952583

    Sample: Tissue homogenate
  26. Chronic Diseases Prevention Review (2019)
    Protective effect of Alginate oligosaccharide on senescent cardiomyocytes

    DOI:

    PMID: 99999999

    Sample: Cell lysate
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