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Signal Transduction Protein Phosphorylation Tyrosine Kinases Receptor Tyrosine Kinases

Anti-Insulin Receptor (phospho Y972)抗体(ab5678)

  • Datasheet
  • SDS
Reviews (2)Q&A (4)References (8)

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Immunocytochemistry/ Immunofluorescence - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)
  • Western blot - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)

Key features and details

  • Rabbit polyclonal to Insulin Receptor (phospho Y972)
  • Suitable for: ICC/IF, WB
  • Reacts with: Human
  • Isotype: IgG

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概述

  • 产品名称

    Anti-Insulin Receptor (phospho Y972)抗体
    参阅全部 Insulin Receptor 一抗
  • 描述

    兔多克隆抗体to Insulin Receptor (phospho Y972)
  • 宿主

    Rabbit
  • 特异性

    In some cell systems ab5678 has been shown to cross-react with IGF1R pY950 (75% homologous).
  • 经测试应用

    适用于: ICC/IF, WBmore details
  • 种属反应性

    与反应: Human
    预测可用于: Mouse, Rat
  • 免疫原

    Synthetic peptide corresponding to Human Insulin Receptor (phospho Y972).

  • 阳性对照

    • IF: Insulin treated MCF7 cells. WB: CHO-T (Chinese hamster ovary cell line) cells transfected with a vector encoding the human insulin receptor and stimulated with insulin.
  • 常规说明

    Biological actions of insulin are mediated by the Insulin Receptor (IR), a receptor tyrosine kinase that regulates multiple signaling pathways through activation of a series of phosphorylation cascades. The IR is a heterotetrameric protein consisting of two ligand-binding alpha subunits and two beta subunits that each contain a tyrosine kinase domain. Insulin binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Tyrosine 972 is in the juxtamembrane Asn-Pro- Glu-Tyr (NPEY) motif. Phosphorylation of IR tyrosine 972 is required for the binding and/or phosphorylation of the adapter protein Shc, the PTB domain, IRS-1, PI3 kinase, and the Suppressor of Cytokine Signaling (SOCS).

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

性能

  • 形式

    Liquid
  • 存放说明

    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.
  • 存储溶液

    pH: 7.30
    Preservative: 0.05% Sodium azide
    Constituents: PBS, 1% BSA, 50% Glycerol
  • Concentration information loading...
  • 纯度

    Immunogen affinity purified
  • 纯化说明

    The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Insulin Receptor (IR). The final product is generated by affinity chromatography using an IR-derived peptide phosphorylated at tyrosine 972.
  • Primary antibody说明

    Biological actions of insulin are mediated by the Insulin Receptor (IR), a receptor tyrosine kinase that regulates multiple signaling pathways through activation of a series of phosphorylation cascades. The IR is a heterotetrameric protein consisting of two ligand-binding alpha subunits and two beta subunits that each contain a tyrosine kinase domain. Insulin binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Tyrosine 972 is in the juxtamembrane Asn-Pro- Glu-Tyr (NPEY) motif. Phosphorylation of IR tyrosine 972 is required for the binding and/or phosphorylation of the adapter protein Shc, the PTB domain, IRS-1, PI3 kinase, and the Suppressor of Cytokine Signaling (SOCS).
  • 克隆

    多克隆
  • 同种型

    IgG
  • 研究领域

    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Receptor Tyrosine Kinases
    • Signal Transduction
    • Growth Factors/Hormones
    • Insulin / Insulin-like
    • Neuroscience
    • Neurology process
    • Metabolism
    • Cancer
    • Growth factors
    • Insulin and insulin-like
    • Cardiovascular
    • Atherosclerosis
    • Diabetes associated
    • Metabolism
    • Types of disease
    • Diabetes
    • Metabolism
    • Types of disease
    • Cancer
    • Metabolism
    • Types of disease
    • Heart disease

相关产品

  • Affibody® Molecule

    • Anti-Insulin Affibody® Molecule (ab31906)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
  • Recombinant Protein

    • Recombinant human Insulin Receptor protein (ab80251)

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab5678于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
ICC/IF
1/100 - 1/500.
WB (2)
1/1000. Detects a band of approximately 110 kDa.
说明
ICC/IF
1/100 - 1/500.
WB
1/1000. Detects a band of approximately 110 kDa.

靶标

  • 功能

    Receptor tyrosine kinase which mediates the pleiotropic actions of insulin. Binding of insulin leads to phosphorylation of several intracellular substrates, including, insulin receptor substrates (IRS1, 2, 3, 4), SHC, GAB1, CBL and other signaling intermediates. Each of these phosphorylated proteins serve as docking proteins for other signaling proteins that contain Src-homology-2 domains (SH2 domain) that specifically recognize different phosphotyrosines residues, including the p85 regulatory subunit of PI3K and SHP2. Phosphorylation of IRSs proteins lead to the activation of two main signaling pathways: the PI3K-AKT/PKB pathway, which is responsible for most of the metabolic actions of insulin, and the Ras-MAPK pathway, which regulates expression of some genes and cooperates with the PI3K pathway to control cell growth and differentiation. Binding of the SH2 domains of PI3K to phosphotyrosines on IRS1 leads to the activation of PI3K and the generation of phosphatidylinositol-(3, 4, 5)-triphosphate (PIP3), a lipid second messenger, which activates several PIP3-dependent serine/threonine kinases, such as PDPK1 and subsequently AKT/PKB. The net effect of this pathway is to produce a translocation of the glucose transporter SLC2A4/GLUT4 from cytoplasmic vesicles to the cell membrane to facilitate glucose transport. Moreover, upon insulin stimulation, activated AKT/PKB is responsible for: anti-apoptotic effect of insulin by inducing phosphorylation of BAD; regulates the expression of gluconeogenic and lipogenic enzymes by controlling the activity of the winged helix or forkhead (FOX) class of transcription factors. Another pathway regulated by PI3K-AKT/PKB activation is mTORC1 signaling pathway which regulates cell growth and metabolism and integrates signals from insulin. AKT mediates insulin-stimulated protein synthesis by phosphorylating TSC2 thereby activating mTORC1 pathway. The Ras/RAF/MAP2K/MAPK pathway is mainly involved in mediating cell growth, survival and cellular differentiation of insulin. Phosphorylated IRS1 recruits GRB2/SOS complex, which triggers the activation of the Ras/RAF/MAP2K/MAPK pathway. In addition to binding insulin, the insulin receptor can bind insulin-like growth factors (IGFI and IGFII). Isoform Short has a higher affinity for IGFII binding. When present in a hybrid receptor with IGF1R, binds IGF1. PubMed:12138094 shows that hybrid receptors composed of IGF1R and INSR isoform Long are activated with a high affinity by IGF1, with low affinity by IGF2 and not significantly activated by insulin, and that hybrid receptors composed of IGF1R and INSR isoform Short are activated by IGF1, IGF2 and insulin. In contrast, PubMed:16831875 shows that hybrid receptors composed of IGF1R and INSR isoform Long and hybrid receptors composed of IGF1R and INSR isoform Short have similar binding characteristics, both bind IGF1 and have a low affinity for insulin.
  • 组织特异性

    Isoform Long and isoform Short are predominantly expressed in tissue targets of insulin metabolic effects: liver, adipose tissue and skeletal muscle but are also expressed in the peripheral nerve, kidney, pulmonary alveoli, pancreatic acini, placenta vascular endothelium, fibroblasts, monocytes, granulocytes, erythrocytes and skin. Isoform Short is preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney. Found as a hybrid receptor with IGF1R in muscle, heart, kidney, adipose tissue, skeletal muscle, hepatoma, fibroblasts, spleen and placenta (at protein level). Overexpressed in several tumors, including breast, colon, lung, ovary, and thyroid carcinomas.
  • 疾病相关

    Rabson-Mendenhall syndrome
    Leprechaunism
    Diabetes mellitus, non-insulin-dependent
    Familial hyperinsulinemic hypoglycemia 5
    Insulin-resistant diabetes mellitus with acanthosis nigricans type A
  • 序列相似性

    Belongs to the protein kinase superfamily. Tyr protein kinase family. Insulin receptor subfamily.
    Contains 3 fibronectin type-III domains.
    Contains 1 protein kinase domain.
  • 结构域

    The tetrameric insulin receptor binds insulin via non-identical regions from two alpha chains, primarily via the C-terminal region of the first INSR alpha chain. Residues from the leucine-rich N-terminus of the other INSR alpha chain also contribute to this insulin binding site. A secondary insulin-binding site is formed by residues at the junction of fibronectin type-III domain 1 and 2.
  • 翻译后修饰

    After being transported from the endoplasmic reticulum to the Golgi apparatus, the single glycosylated precursor is further glycosylated and then cleaved, followed by its transport to the plasma membrane.
    Autophosphorylated on tyrosine residues in response to insulin. Phosphorylation of Tyr-999 is required for binding to IRS1, SHC1 and STAT5B. Dephosphorylated by PTPRE at Tyr-999, Tyr-1185, Tyr-1189 and Tyr-1190. Dephosphorylated by PTPRF and PTPN1. Dephosphorylated by PTPN2; down-regulates insulin-induced signaling.
  • 细胞定位

    Cell membrane.
  • Target information above from: UniProt accession P06213 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 数据库链接

    • Entrez Gene: 3643 Human
    • Entrez Gene: 16337 Mouse
    • Entrez Gene: 24954 Rat
    • Omim: 147670 Human
    • SwissProt: P06213 Human
    • SwissProt: P15208 Mouse
    • SwissProt: P15127 Rat
    • Unigene: 465744 Human
    • Unigene: 268003 Mouse
    • Unigene: 9876 Rat
    see all
  • 别名

    • CD220 antibody
    • HHF5 antibody
    • human insulin receptor antibody
    • Insr antibody
    • INSR_HUMAN antibody
    • Insulin receptor subunit beta antibody
    • IR 1 antibody
    • IR antibody
    • IR-1 antibody
    • IR1 antibody
    see all

图片

  • Immunocytochemistry/ Immunofluorescence - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)
    Immunocytochemistry/ Immunofluorescence - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)

    Immunofluorescence analysis of insulin treated MCF7 cells labelling Insulin Receptor (phospho Y972) (Panel a: green) using ab5678 at 2µg/mL in 1% BSA for 3 hours at room temperature, followed by Alexa Fluor 488® Goat Anti-Rabbit IgG Secondary Antibody at 1/400 dilution for 30 minutes at room temperature. Panel b:Nuclei were stained with DAPI (blue). Panel c:  F-actin was stained with Alexa Fluor 594® Phalloidin (red). Panel d: Merged image showing membrane localization. Panel e: Untreated MCF7 cells. Panel f: Control, no primary antibodyl. The images were captured at 20X magnification.

    Prior antibody incubation, MCF7 (human breast adenocarcinoma cell line) cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature, followed by treatment with 100nM of insulin for 5 min. Assay was done on 70% confluent log phase MCF7 cells.

     

  • Western blot - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)
    Western blot - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)
    All lanes : Anti-Insulin Receptor (phospho Y972) antibody (ab5678) at 1/1000 dilution (2 hours at room temperature in a 3% BSA-TBST buffer)

    Lane 1 : Unstimulated (-), CHO-T transfected with insulin receptor containing vector whole cell extract with 5% BSA-TBST buffer for one hour at room temperature
    Lanes 2-5 : Stimulated (+) with 50 nM insulin for 5 minutes, CHO-T transfected with insulin receptor containing vector whole cell extract with 5% BSA-TBST buffer for one hour at room temperature

    Secondary
    All lanes : Goat F (ab')2 anti-rabbit IgG HRP conjugate


    Upregulation and Antibody-Peptide Competition:

    Prior primary antibody incubation:

    1 and 2 - no peptide;

    3 -  non-phosphorylated peptide corresponding to the phosphopeptide immunogen;

    4 - generic phosphotyrosine-containing peptide;

    5 - phosphopeptide immunogen.

    SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF.

    The data show that only the phosphopeptide corresponding to ab5678 completely blocks the antibody signal, demonstrating the specificity of the antibody.

    The data also show up-regulation of the signal upon stimulation with insulin in this cell system.

实验方案

  • Western blot protocols
  • Immunoprecipitation protocols
  • Immunocytochemistry & immunofluorescence protocols

Click here to view the general protocols

数据表及文件

  • SDS download

  • Datasheet download

    Download

文献 (8)

发表研究结果有使用 ab5678?请让我们知道,以便我们可以引用本数据表中的参考文章。

ab5678 被引用在 8 文献中.

  • Gong M  et al. Wenxin Keli Regulates Mitochondrial Oxidative Stress and Homeostasis and Improves Atrial Remodeling in Diabetic Rats. Oxid Med Cell Longev 2020:2468031 (2020). PubMed: 32104528
  • Nielsen TL  et al. Exercising with blocked muscle glycogenolysis: Adaptation in the McArdle mouse. Mol Genet Metab 123:21-27 (2018). WB . PubMed: 29174367
  • Blesson CS  et al. Gestational Protein Restriction Impairs Glucose Disposal in the Gastrocnemius Muscles of Female Rats. Endocrinology 158:756-767 (2017). PubMed: 28324067
  • Krüger J  et al. Enhanced insulin signaling in density-enhanced phosphatase-1 (DEP-1) knockout mice. Mol Metab 4:325-36 (2015). WB . PubMed: 25830095
  • Catalano KJ  et al. Insulin resistance induced by hyperinsulinemia coincides with a persistent alteration at the insulin receptor tyrosine kinase domain. PLoS One 9:e108693 (2014). WB . PubMed: 25259572
  • Blesson CS  et al. Gestational protein restriction impairs insulin-regulated glucose transport mechanisms in gastrocnemius muscles of adult male offspring. Endocrinology 155:3036-46 (2014). WB ; Rat . PubMed: 24797633
  • Storey SM  et al. Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes. Am J Physiol Gastrointest Liver Physiol 303:G837-50 (2012). PubMed: 22859366
  • Uhles S  et al. Selective gene activation by spatial segregation of insulin receptor B signaling. FASEB J 21:1609-21 (2007). WB, IP ; Rat . PubMed: 17264162

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1-6 of 6 Abreviews or Q&A

Western blot abreview for Anti-Insulin Receptor (phospho Y972) antibody

Poor
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Chinese hamster Cell lysate - whole cell (CHO Expressing Human Insulin Receptor Þ)
Gel Running Conditions
Reduced Denaturing (Biorad TGX Stain-Free, 10%)
Loading amount
30 µg
Treatment
10 nM Insulin for 10min
Specification
CHO Expressing Human Insulin Receptor Þ
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Dr. yang lou

Verified customer

提交于 Jul 17 2017

Western blot abreview for Anti-Insulin Receptor (phospho Y972) antibody

Average
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Mouse Tissue lysate - whole (Melanoma cancer)
Loading amount
30 µg
Specification
Melanoma cancer
Treatment
5gy Irradiation
Gel Running Conditions
Non-reduced Denaturing (8%)
Blocking step
Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 3% · Temperature: 25°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Ms. Seontae Kim

Verified customer

提交于 Jan 14 2013

Question

Can I use Goat anti-rabbit IgG as a secondary antibody instead of Goat anti-rabbit (Fab)2' as described in data sheet with ab5678? Thanks

Read More

Abcam community

Verified customer

Asked on Feb 11 2013

Answer



I am happy to confirm that goat anti-rabbit IgG can be used a secondary with ab5678.

Read More

Abcam Scientific Support

回复于 Feb 11 2013

Question

No signal detected in western blots of insulin-treated controls or experimental samples.

Read More

Abcam community

Verified customer

Asked on Oct 06 2011

Answer

Thank you for contacting us about the issue you are having with ab5678. I noticed after we spoke that the datasheet for the antibody I proposed sending, the rabbit monoclonal anti-Insulin Receptor (phospho Y1185), has a note stating that it will not react with mouse samples. I went back to the list we were studying and found another that may be a better choice: Insulin Receptor (phospho Y1158) antibody (ab78355) Click here (or use the following: https://www.abcam.com/Insulin-Receptor-phospho-Y1158-antibody-ab78355.html). The antibody has not been tested against mouse samples but is raised against a sequence conserved in mouse. I will offer our guarantee for this antibody, in case it does not work for you. We usually only offer a guarantee for tested species and applications, but I am fairly confident this will be able to detect the phosphorylation in mouse samples. Please let me know if you would like to try this, ab78355, instead of the rabbit monoclonal, which we will not guarantee, given the negative data for mouse. The antibody is in stock and available for overnight delivery.

Read More

Abcam Scientific Support

回复于 Oct 06 2011

Question

Does this antibody recognize the alpha subunit or the beta subunit?

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Abcam community

Verified customer

Asked on Apr 19 2006

Answer

Thank you for your enquiry. The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of the human insulin receptor that contains tyrosine 972, as numbered according to Ebina, et al. (tyrosine 960 according to Ullrich, et al.) Tyrosine 972 is in the juxtamembrane Asn-Pro-Glu-Tyr (NPEY) motif of the B subunit. Please contact us again if you have any additional questions.

Read More

Abcam Scientific Support

回复于 Apr 20 2006

Question

BATCH NUMBER 99451 ORDER NUMBER V06053 DESCRIPTION OF THE PROBLEM Non-specific band, wrong band size. SAMPLE Cell extract. PRIMARY ANTIBODY Primary antibody is used here ab5678 and at the dilution at 0.5ug/ml in 5% TBS milk for 2hrs. at RT. DETECTION METHOD ECL for 2mins. POSITIVE AND NEGATIVE CONTROLS USED No positive and negative control is used here. ANTIBODY STORAGE CONDITIONS After opening the pack, the antibody is stored at -20C SAMPLE PREPARATION In the lysis buffer, protease inhibitors are already added.And once the sample is prepared, after adding 4X sample buffer,it is to be heated upto 100C for 5 mins. AMOUNT OF PROTEIN LOADED In microgram amount but exactly the OD in spectrophotometer at 595nm, is arround 0.4. ELECTROPHORESIS/GEL CONDITIONS Reducing gel and 10% sds gel. TRANSFER AND BLOCKING CONDITIONS 1.Transfer buffer is used as per the protocol and over night transfer on nitrocellulose membrane. 2. 5%TBS milk is used for blocking the membrane after the transfer for one hour at room temperature(RT). SECONDARY ANTIBODY Secondary is used Goat-Anti-Rabbit (1:1000) from [a competitor] with 5%TBS milk for one hour at RT. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? No steps. ADDITIONAL NOTES Nothing at this point but want to know the your at the earliest and all the possible points to get my result back.

Read More

Abcam community

Verified customer

Asked on May 12 2005

Answer

I'm sorry to hear you are having a problem with ab5678. It would be helpful if you could also tell me the type of cells you are using, what size bands you see, and you could also attach a picture of your blot and that would be very helpful. Using the information you have provided to me so far, I would like to suggest the following modifications to your protocol: 1) a positive and negative control would be very helpful such as cells transfected with human insulin receptor +/- insulin treatment, 2) try varying the concentration of antibody and using it at 4 degrees overnight, 3) remove the milk from the primary and secondary, and then if there are too many bands add milk to 0.5 - 1%, 4) figure out the amount of protein you are adding and load 20 to 30 ug protein per lane, 5) try using the secondary at 1:10,000. Please let me know if this helps and do not hesitate to contact us for further advice,

Read More

Abcam Scientific Support

回复于 May 13 2005

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