Anti-LGR5 antibody (ab75732)

Rabbit polyclonal LGR5 antibody. Validated in WB, ELISA, IHC, Flow Cyt, ICC/IF and tested in Mouse, Human. Cited in 23 publication(s). Independently reviewed in 15 review(s).

Overview

  • Product name
  • Description
    Rabbit polyclonal to LGR5
  • Host species
    Rabbit
  • Specificity
    LGR5 is a multi-pass membrane protein, due to hydrophobic nature these proteins tend to aggregate when boiled at 100°C in western blotting therefore, we recommend slow heating of lysates for 15-20 minutes at 60-70°C rather than boiling. The immunogen used for this product shares 62% homology with LGR6. Cross-reactivity with this protein has not been confirmed experimentally.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, Flow Cyt, WB, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Cow
  • Immunogen

    Synthetic peptide within Human LGR5 aa 689-719 conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
    Database link: O75473
    (Peptide available as ab185636)

  • Positive control
    • WB: Mouse cerebellum tissue lysate. IHC-P: Human brain tissue. ICC/IF: HeLa cells. Flow Cyt: HepG2 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab75732 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
ICC/IF 1/100.
IHC-P 1/10 - 1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Please note for better results the antigen retrieval method may need optimizations. We recommend trying a range of incubation time e.g. 1, 2, 3, 4, 5 minutes using pressure cooker or 3, 5, 10, 15 minutes in microwave after buffer start boiling. For more details check IHC-P – detailed guide in protocol section.

Flow Cyt 1/10 - 1/50.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

WB 1/1000 - 1/2000. Detects a band of approximately 100 kDa (predicted molecular weight: 100 kDa).

Blocking peptide protocol

ELISA 1/1000.

Target

  • Function
    Orphan receptor. Stem cell marker of the intestinal epithelium and the hair follicule. Target gene of Wnt signaling.
  • Tissue specificity
    Expressed in skeletal muscle, placenta, spinal cord, and various region of brain. Expressed at the base of crypts in colonic and small mucosa stem cells. In premalignant cancer expression is not restricted to the cript base. Overexpressed in cancers of the ovary, colon and liver.
  • Sequence similarities
    Belongs to the G-protein coupled receptor 1 family.
    Contains 16 LRR (leucine-rich) repeats.
    Contains 1 LRRNT domain.
  • Cellular localization
    Cell membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • FEX antibody
    • G protein coupled receptor 49 antibody
    • G protein coupled receptor 67 antibody
    • g protein-coupled receptor fex antibody
    • G-protein coupled receptor 49 antibody
    • G-protein coupled receptor 67 antibody
    • G-protein coupled receptor HG38 antibody
    • GPCR GPR49 antibody
    • GPR 49 antibody
    • GPR 67 antibody
    • GPR49 antibody
    • GPR67 antibody
    • GRP 49 antibody
    • GRP49 antibody
    • HG 38 antibody
    • HG38 antibody
    • Leucine rich repeat containing G protein coupled receptor 5 antibody
    • Leucine-rich repeat-containing G-protein coupled receptor 5 antibody
    • LGR 5 antibody
    • LGR5 antibody
    • LGR5_HUMAN antibody
    • MGC117008 antibody
    • Orphan G protein coupled receptor HG38 antibody
    see all

Images

  • Immunohistochemical analysis of PFA fixed, paraffin-embedded human brain tissue sections labelling LGR5 with ab75732 at 1/25 dilution.

  • All lanes : Anti-LGR5 antibody (ab75732) at 1/2000 dilution

    Lane 1 : Mouse skeletal muscle lysates
    Lane 2 : Rat skeletal muscle lysates
    Lane 3 : Hela whole cell lysates
    Lane 4 : HepG2 whole cell lysates
    Lane 5 : SH-SY5Y whole cell lysates

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution

    Predicted band size: 100 kDa



    Blocking buffer 5% NFDM/TBST

    Diluting buffer 5% NFDM/TBST

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling LGR5 with ab75732. Cells were fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min) and then incubated with the primary antibody (1/100, 2 h at room temperature). An Alexa Fluor® 488 conjugated donkey anti-rabbit antibody (green) was used as the secondary antibody (1/1000, 1h). Nuclei were counterstained with Hoechst 33342 (blue) (10 μg/ml, 5 min).

  • Overlay histogram showing 2% paraformaldehyde fixed SH-SY5Y cells stained with ab75732 (green line). Cells were incubated with the antibody at 1/25 dilution at 37oC for 60 minutes. The secondary antibody used was DyLight® 488 Goat-Anti-Rabbit IgG at 1/400 dilution. A non-specific IgG antibody was used for an isotype control (blue line).

  • Immunohistochemical analysis of PFA fixed, paraffin-embedded mouse brain tissue sections labelling LGR5 with ab75732 at 1/25 dilution.

  • Anti-LGR5 antibody (ab75732) at 1/750 dilution (for 16 hours at 4°C) + Whole tissue lysate of mouse brain at 18 µg

    Secondary
    An HRP-conjugated Goat polyclonal at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 100 kDa
    Observed band size: 97 kDa
    why is the actual band size different from the predicted?


    Exposure time: 2 minutes


    Blocking Step: 5% Milk for 1 hour at 20°C.

    See Abreview

  • ab75732 at 0.025 mg/ml staining LGR5 in human Hela cells by Immunocytochemistry / Immunofluorescence.

  • Overlay histogram showing mouse intestinal cells stained with ab75732 (pink line). Cells were incubated with the antibody at 1/50 in DMEM at 20°C for 30 minutes.The secondary antibody used was DyLight® 649 goat anti-rabbit IgG at 1/100 dilution. A non-specific IgG antibody was used for an isotype control (black line).

    See Abreview

References

This product has been referenced in:
  • Chen Z & Xue C G-Protein-Coupled Receptor 5 (LGR5) Overexpression Activates ß-Catenin Signaling in Breast Cancer Cells via Protein Kinase A. Med Sci Monit Basic Res 25:15-25 (2019). Read more (PubMed: 30662060) »
  • López-Arribillaga E  et al. Manic Fringe deficiency imposes Jagged1 addiction to intestinal tumor cells. Nat Commun 9:2992 (2018). Read more (PubMed: 30065304) »
See all 25 Publications for this product

Customer reviews and Q&As

1-10 of 34 Abreviews or Q&A

Answer

Thanks for your reply.

I am sorry to hear some of your colleagues have had a bad experience regarding our "Abpromise". I would encourage them to contact us to resolve any issues they have.

The Abpromise landing page to which I sent you a link does have quite a bit of text on it and its easy to miss the key points regarding your question. Below I have pasted these relevant items from the Abpromise page:

Replacement or refund for products not performing as stated on the datasheet
"Predicted to react" information on datasheets is based on sequence homology and provided for reference only
"Predicted to react" species are not guaranteed, as they have not been tested



To expand on the above, we guarantee the antibodies to work in the applications listed in the 'Tested application' section and to react with the species listed in the 'Cross reactivity' section.

How does the Abpromise apply to ab24386?:
We guarantee it to work in IHC-P and to react with Human LRP6 protein containing the epitope CMTSVATAKGYTSDL, corresponding to amino acids 1546-1560 of Human LRP6.

Protein orthologs that contain a region which has at least 95% identity to the above epitope, but have not yet been tested for reactivity, are listed in the 'Predicted to work with' section. For ab24386, these species include Mouse, Rat, Chicken, Dog, Chimpanzee. As noted above, "Predicted to react" species are not guaranteed, as they have not been tested.

How does the Abpromise apply to ab75732?:
We guarantee it to work in ICC/IF, IHC-P, Flow Cyt, WB, ELISA andICCand to react with Human and MouseLRP6 protein containing an epitope found within amino acids 700-730 of the human protein.

What does the guarantee mean for you:
If, after doing your experiment, the antibody does not appear to work as expected (ie. as noted above), we ask you to contact us within 6 months of receiving the antibody. At this point we will offer troubleshooting tipsand may ask further questions to help determine if it is the antibody or perhaps another aspect of the experiment that is the issue. If in the end it does appear that the antibody is the problem, we are happy to offer a replacement with any primary antibody, credit for a future purchase or refund.

I hope this is helpful. Please contact me if you have any further questions.

Read More

Answer

Thank you for contacting us. Below is the protocol that was used: Preparing Tissue Sections for Immunostaining: • Fix the tissue in 10% formalin at 4°C overnight. • Embed fixed tissue in paraffin. • Mount tissue sections on slides. • Clear the paraffin with xylene for ten minutes; move slides to a fresh dish of xylene for an additional ten minutes. NOTE: Perform all xylene washes in a fume hood! • Rinse the slides twice for 2 minutes in 100% alcohols (18:1:1 100% ethanol:100% methanol:100% isopropanol). • Rinse the slides twice for 2 minutes in a 95% solution of the 100% alcohols. • Place slides in an 80% solution of the 100% alcohols for 2 minutes, followed by deionized water for 5 minutes. • Rinse slides several times with fresh deionized water followed by another five minutes wash using fresh water. Sodium Citrate Antigen Retrieval: • Place slides in a glass slide holder and fill in the rest of the rack with blank slides (10 total) to ensure even heating. • Place rack in 600 ml of 10 mM sodium citrate, pH 6.0 in a glass 2L-beaker. Mark a line at the top of the liquid on the beaker. • Microwave for 20 min total, replacing evaporated water every 5 min. • Cool slides for 20 min. • Wash 4 x 3 min in ddH2O, and 3 min in 1X PBS. Blocking • Block endogenous peroxidases by soaking slides in a solution of 90% methanol/3% H2O2 for 15 minutes at room temperature. Wash 3X in PBS. • Immerse slides in a dish containing blocking buffer (serum from host species of secondary antibody to be used, diluted 1:10 in TBS). Incubate at 37°C for one hour. Incubation with Primary Antibodies • Cover the tissue sections with primary antibody diluted in blocking buffer. Antibody is diluted 1:50 and 1:100. Incubate for 1 hour at 37°C. • Blot excess liquid from slides and rinse three times in PBS for five minutes each wash. Incubation with Secondary Antibodies • Cover the tissue sections with secondary antibody diluted in blocking buffer according to manufacturer’s instructions. We routinely use prediluted universal secondary antibody. Incubate at 37°C for 30 min. • Blot excess liquid and rinse twice in TBS for five minutes each wash. Counterstaining and Visualization • Counterstain with Hematoxylin. • Rinse several times in deionized water. Blot excess water around tissue, then apply one drop of mounting media to tissue and place coverslip over slide. Seal with nail polish. Citrate Solutions: Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate based solution is designed to break the protein cross-links, thereby unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections and enhancing the staining intensity of antibodies. Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0): Tri-sodium citrate (dihydrate) ------- 2.94 g Distilled water --------------------------- 1000 ml Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 °C for longer storage. Washing Buffer: 1xPBS: NaCl --------------------------------------- 8 g KCl ----------------------------------------- 0.2 g Na2HPO4----------------------------------1.44 g KH2PO4------------------------------------0.24 g Distilled Water---------------------------800 mL Adjust pH to 7.2 with HCl. Adjust volume to 1 L with additional H2O Normal Serum Blocking Buffer: 2% serum from host species of secondary antibody (blocking) 1% BSA (stabilizer) 0.1% cold fish skin gelatin (blocking) 0.1% Triton X-100 (penetration enhancer) 0.05% Tween 20 (detergent and surface tension reducer) 0.05% sodium azide (preservative) Dissolve in 1xPBS Mix well and store at 4 ºC. Avidin/Biotin Block: Avidin 0.001% in 1xPBS Biotin 0.001% in 1xPBS Store these blocking solution at 4 ºC. Primary Antibody Dilution Buffer: 1%BSA (stabilizer and blocking) 0.1% cold fish skin gelatin (blocking) 0.05% sodium azide (preservative) 0.01M PBS pH7.2 Note: 1) Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. 2) This buffer cannot be used for diluting HRP conjugated antibodies since sodium azide is an inhibitor of HRP. Peroxidase Blocking Solution (3% H2O2 in PBS): 30% H2O2 -------------------------- 2 ml 1X PBS ----------------------------- 18 ml Mix well and store at 4 ºC for up to 3 months. This solution is recommended for paraffin sections I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (adult ilium)
Permeabilization
No
Specification
adult ilium
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Methanol

Abcam user community

Verified customer

Submitted Dec 04 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Monkey Tissue sections (Colon and Jejunum)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate acid
Permeabilization
Yes - Tween-20/Triton X 100
Specification
Colon and Jejunum
Blocking step
Casein-based blocking solution (Blocking sniper-Biocare medical) as blocking agent for 8 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Paraformaldehyde

Dr. Li Li

Verified customer

Submitted Mar 13 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Colon)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer
Permeabilization
No
Specification
Colon
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 21 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Bos taurus Tissue sections (Airway)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris-EDTA, pH9.0
Permeabilization
No
Specification
Airway
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 2.5% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Dec 23 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step
None
Sample
Human Tissue sections (tooth)
Specification
tooth
Permeabilization
No
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jul 07 2014

Application
Flow Cytometry
Fixation
none
Permeabilization
No
Sample
Mouse Cell (intestinal cells)
Specification
intestinal cells
Gating Strategy
high FSC/SSC popn log scale
Preparation
Cell harvesting/tissue preparation method: isolate colon, collagenase digestion
Sample buffer: DMEM

Abcam user community

Verified customer

Submitted Aug 20 2013

Answer

While we do not have specific data regarding this products use in FACS sorting, the immunogen area is within an extracellular region. As such, it seems very likely that this antibody will be successful in that application.

Read More

Answer

Thank you for the clarification.
You are right, this antibody is indeed tested in Flow, but with a conjugated secondary antibody.
If you would like to use this antibody conjugated directly to a fluorochrome, I would suggest to have a look at our conjugation and purification kits:
https://www.abcam.com/index.html?datasheet=102776 https://www.abcam.com/index.html?datasheet=102776.
As an example, the FITC labelling kit: https://www.abcam.com/index.html?datasheet=102885 https://www.abcam.com/index.html?datasheet=102885.
For more information and to get an overview about the working mechanism of this kits, please see the PDF I have attached to this email.

Read More

1-10 of 34 Abreviews or Q&A

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