Overview

  • Product name

    Anti-GPA33 antibody [EPR4240]
    See all GPA33 primary antibodies
  • Description

    Rabbit monoclonal [EPR4240] to GPA33
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IP, IHC-Pmore details
    Unsuitable for: Flow Cyt or ICC
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human GPA33 aa 300-400. The exact sequence is proprietary.
    Database link: Q99795

  • Positive control

    • HT-29, Human small intestine, and Human fetal colon lysates. Human colon tissue. Human colonic adenocarcinoma tissue.
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab108938 in the following tested applications.

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

Application Abreviews Notes
WB 1/1000 - 1/10000. Predicted molecular weight: 36 kDa.
IP 1/10 - 1/100.
IHC-P 1/250 - 1/500. Antigen retrieval recommended; heat up to 98 degrees C, below boiling, and then let cool for 10-20 min.
  • Application notes
    Is unsuitable for Flow Cyt or ICC.
  • Target

    • Function

      May play a role in cell-cell recognition and signaling.
    • Tissue specificity

      Expressed in normal gastrointestinal epithelium and in 95% of colon cancers.
    • Sequence similarities

      Contains 1 Ig-like C2-type (immunoglobulin-like) domain.
      Contains 1 Ig-like V-type (immunoglobulin-like) domain.
    • Post-translational
      modifications

      N-glycosylated, contains approximately 8 kDa of N-linked carbohydrate.
      Palmitoylated.
    • Cellular localization

      Membrane.
    • Information by UniProt
    • Database links

    • Alternative names

      • A33 antibody
      • Cell surface A33 antigen antibody
      • Cell surface A33 antigen precursor antibody
      • Glycoprotein A33 (transmembrane) antibody
      • Glycoprotein A33 antibody
      • Gpa33 antibody
      • GPA33_HUMAN antibody
      • MGC129986 antibody
      • MGC129987 antibody
      • OTTHUMP00000032346 antibody
      • Transmembrane glycoprotein A33 antibody
      see all

    Images

    • All lanes : Anti-GPA33 antibody [EPR4240] (ab108938) at 1/1000 dilution

      Lane 1 : HT-29 lysate
      Lane 2 : Human small intestine lysate
      Lane 3 : Human fetal colon lysates

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP-labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 36 kDa

    • ab108938 at 1/250 dilution staining GPA33 in paraffin-embedded Human colon tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
    • ab108938 at 1/250 dilution staining GPA33 in paraffin-embedded Human colonic adenocarcinoma tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
    • ab108938 showing negative staining in Breast carcinoma tissue.

    • ab108938 showing negative staining in Thyroid gland carcinoma tissue.

    • ab108938 showing positive staining in Ovarian carcinoma tissue.

    • ab108938 showing negative staining in Normal tonsil tissue.

    • ab108938 showing negative staining in Normal liver tissue.

    References

    This product has been referenced in:

    • Gotanda K  et al. Circulating intestine-derived exosomal miR-328 in plasma, a possible biomarker for estimating BCRP function in the human intestines. Sci Rep 6:32299 (2016). WB, IP . Read more (PubMed: 27571936) »
    See 1 Publication for this product

    Customer reviews and Q&As

    1-3 of 3 Abreviews or Q&A

    Application
    Western blot
    Sample
    Human Cell lysate - other (Colorectal Cancer Cell Lines and Exosomes)
    Gel Running Conditions
    Reduced Denaturing (4- 20%)
    Loading amount
    40 µg
    Specification
    Colorectal Cancer Cell Lines and Exosomes
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

    Fabia Fricke

    Verified customer

    Submitted Dec 19 2017

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Colon tissue)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citrat
    Permeabilization
    No
    Specification
    Colon tissue
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 20°C
    Fixative
    Formaldehyde

    Fabia Fricke

    Verified customer

    Submitted Dec 18 2017

    Answer

    Thank you for contacting us. We do not know of any publications using ab67102 or ab108938. The protocol used with ab67102 is below: Blocking buffer (also Dilution buffer) Weigh non-fat milk 5 g and dissolve in 100 mL 1X PBST (0.2%) to a final mixture of 5% non-fat milk/PBST (0.2%).  (10X) PBST (phosphate buffer saline) NaCl (0.13 M x 10, Merck 6404) 75.9 g NaH2PO4·H2O (0.01 M x 10, Merck A429146 335) 13.8 g Add 800 mL ddH2O. After the salts have dissolved, use NaOH liquid to adjust the solution to pH 7.0 and make the final dilute solution to 1,000 mL. The solution becomes 10X PBS. Dilute the solution with ddH2O to the final 1X PBS prior application. PBST (0.2%) (Phosphate buffer saline and 0.2% Tween 20) 1X PBS 1 L Tween 20 2 mL Dilute 10X PBS with ddH2O to a final 1X concentration. Add Tween 20 [0.2% (v/v)] to final PBST (0.2%).   Anti-IgG Secondary antibody Goat Anti-Mouse IgG (H&L)-HRP Conjugate secondary antibody  Chemiluminescent reagent SuperSignal® West Femto Maximum Sensitivity Substrate (PIERCE, Cat. No. 34094, 34095 & 34096) Reagent must be freshly prepared each time before application. Add adequate amount of blocking buffer and incubate the membrane at room temperature for 1 hour or under 4°C overnight. Dilute the primary antibody with fresh blocking buffer to the designated concentration (1/500). Remove the membrane from the previous blocking buffer and add the diluted primary antibodies to the membrane. Incubate at 4°C overnight. Wash membrane with PBST (0.2%) for 10 minutes. Repeat 3 times. Add adequate amount of anti-IgG secondary antibody. Leave the membrane at room temperature for 1 hour. Wash membrane with PBST (0.2%) for 10 minutes. Repeat 4 times. After washing, place membrane into a sealable bag and add freshly prepared chemiluminescent reagents into the bag to coat the entire membrane. For maximum sensitivity, PIERCE SuperSignal® West Femto Maximum sensitivity substrate is recommended. The reagent should be in 1:1 dilution of reagent A and B as working solution (Strictly follow the provider's instructions). Add working solution to the membrane and seal the bag. Spread the chemiluminescent reagent around so it can be distributed evenly onto the membrane. (Chemiluminescent reagent must be spread out evenly otherwise parts of the membrane will be over-exposed when the photograph is taken.) 0.6 mL of working solution is used for membrane size 8 cm x 10 cm. Take photographs immediately with CCD camera at 5-second, 20-second, 1.5-minute, and 5-minute intervals, in order to acquire proper exposure image.  Strong signals may intensify into blackout signals with hollow band. In this case, dilute the secondary antibody. The protocol for ab52193 is below: 1. Solutions and Reagents 1.1. 10X Cell Lysis Buffer RIPA buffer (Radio Immuno Precipitation Assay buffer) 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 - Add Phenylmethylsulfonyl fluoride (PMSF) toCEll Lysis Buffer to a final concentration of 1mM. (Stock solution 100mM PMSF) NOTE : Add fresh before each use - Add protease and phosphatase inhibitors. - 1X protease inhibitor mixture consists of 2 mM AEBSF, 1 mM EDTA, 130 μM bestatin, 14 μM E-64, and 0.3 μM aprotinin. 1.2. 2X Laemmli Sample Buffer: 62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% Bromophenol Blue, 710 mM beta-mercaptoethanol 1.3. TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) TBST is Tris Base Saline buffer with 0.1% Tween-20 1.5. 5% bovine serum albumin (BSA): Use 25 g in 500 ml TBST 1.6. Goat anti-rabbit HRP antibody 1.7. Chemiluminesence reagents; such as ECL materials and film or imaging system for detection 2. Cell Lysis and Western Blotting Protocol Westerns are performed using cell lysates from harvested cells. 2.1. Adherent cells 2.1.1 Grow cells to ~90% confluency. 2.1.2 Wash cells twice with TBS to remove media 2.1.3 Add the appropriate volume of 1x Cell lysis buffer (ex: 3ml to a T175 flask) 2.1.4 Transfer cell lysates to Eppendorf tubes and sonicate for 10-15 seconds. 2.1.5 Spin at 14,000 rpm, 4 °C for 10 minutes. 2.2. Suspension cells 2.2.1 Pipette cells gently into a conical tube and centrifuge 10 min. at 1000 rpm 2.2.2 Wash cells twice with TBS 2.2.3 Add the appropriate volume of 1x Cell lysis buffer and transfer to Eppendorf tubes (ex: 1ml for 1X10 7 cells) 2.2.4 Sonicate for 10-15 seconds. 2.2.5 Spin at 14,000 rpm, 4 °C for 10 minutes. 2.3. Remove a small volume (50 ul) to perform a protein assay. Determine the protein concentration for each cell lysate. 2.4. To the remaining volume of cell lysate, add an equal volume of 2X Laemmli Sample Buffer (final concentration of 1 mg/ml). 2.5. Boil each cell lysate in sample buffer at 100 °C for 5 min and aliquot. Use a 26-gauge needle to shear released chromatin. Store lysates at -20 °C. Note: Aliquot cell lysates (50-100 μl) in order to avoid repeat freeze/thaw cycles. 2.6. Defrost tubes containing cell lysate at 37 °C. Centrifuge at 14,000 rpm in a microcentrifuge for 5 min. 2.7. Load equal amounts (10-20 μg) cell lysate onto SDS-PAGE gels using gel loading tips, along with molecular weight markers. 2.8. Run gels and transfer for western blotting. 3. Western Blotting 3.1. Block nitrocellulose for 1 hour at room temperature or overnight at 4°C using 5% BSA or 5%. 3.2.Incubate nitrocellulose with appropriate dilutions of primary antibody in 5% BSA overnight at 4°C or for 2 hours at room temperature 3.3. Wash nitrocellulose with three 5-min washes using TBST. 3.4.Incubate nitrocellulose with goat anti-rabbit HRP antibody, diluted to 1:500 to 100,000 in 5% BSA, for 1-2 hours at room temperature. 3.5. Wash nitrocellulose in 3 washes of TBST, then rinse in TBS prior to addition of chemiluminesence reagents. 3.6. Remove excess chemiluminescence reagent and sandwich nitrocellulose blot in any type of transparent plastic wrap (plastic cling wrap, transparent sheet protector, etc.). 3.7. Acquire image using darkroom development techniques. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

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