Overview

  • Product name
    Anti-Lrp2 / Megalin antibody
    See all Lrp2 / Megalin primary antibodies
  • Description
    Rabbit polyclonal to Lrp2 / Megalin
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ELISAmore details
  • Species reactivity
    Reacts with: Recombinant fragment
    Predicted to work with: Human
  • Immunogen

    Synthetic peptide from N-terminus of human LRP2

Applications

Our Abpromise guarantee covers the use of ab56014 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 Use at an assay dependent concentration. Predicted molecular weight: 517 kDa. This antibody has been tested in Western blot against the recombinant peptide used as an immunogen. We have no data on detection of endogenous protein.
ELISA Use at an assay dependent dilution.

Target

  • Function
    Acts together with cubilin to mediate HDL endocytosis (By similarity). May participate in regulation of parathyroid-hormone and para-thyroid-hormone-related protein release.
  • Tissue specificity
    Absorptive epithelia, including renal proximal tubules.
  • Involvement in disease
    Defects in LRP2 are the cause of Donnai-Barrow syndrome (DBS) [MIM:222448]; also known as faciooculoacousticorenal syndrome (FOAR syndrome). DBS is a rare autosomal recessive disorder characterized by major malformations including agenesis of the corpus callosum, congenital diaphragmatic hernia, facial dysmorphology, ocular anomalies, sensorineural hearing loss and developmental delay. The FOAR syndrome was first described as comprising facial anomalies, ocular anomalies, sensorineural hearing loss, and proteinuria. DBS and FOAR were first described as distinct disorders but the classic distinguishing features between the 2 disorders were presence of proteinuria and absence of diaphragmatic hernia and corpus callosum anomalies in FOAR. Early reports noted that the 2 disorders shared many phenotypic features and may be identical. Although there is variability in the expression of some features (e.g. agenesis of the corpus callosum and proteinuria), DBS and FOAR are now considered to represent the same entity.
  • Sequence similarities
    Belongs to the LDLR family.
    Contains 17 EGF-like domains.
    Contains 36 LDL-receptor class A domains.
    Contains 37 LDL-receptor class B repeats.
  • Cellular localization
    Membrane. Membrane > coated pit.
  • Information by UniProt
  • Database links
  • Alternative names
    • Calcium sensor protein antibody
    • DBS antibody
    • Glycoprotein 330 antibody
    • gp330 antibody
    • Heymann nephritis antigen homolog antibody
    • Low-density lipoprotein receptor-related protein 2 antibody
    • LRP-2 antibody
    • Lrp2 antibody
    • LRP2_HUMAN antibody
    • Megalin antibody
    see all

References

ab56014 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (endothelial)
Gel Running Conditions
Reduced Denaturing (6 gel %)
Loading amount
40 µg
Specification
endothelial
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Philip Bilan

Verified customer

Submitted Sep 15 2015

Answer

Thank you for contacting us. I did BLAST searches of our LRP2 antibodies against C.elegans and found that the highest match was with ab56014 with 78%. Usually we like to say that 85% homology between the immunogen and the species will give likely reactivity. This antibody also had a 75% homology with LRP1, so this may not be a good choice for you. All other LRP2 antibodies had lower homology with C. elegans and are not likely to react.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for your enquiry.

We do not have set expiration dates for our products. Most antibodies are stable and can last for anywhere from a few months to several years if stored properly, so we strongly recommend that you follow the storage instructions on the datasheet for the antibody you purchased. These conditions will vary among our antibodies, therefore, it is important to verify the storage conditions for each of our products when you receive them. We guarantee all of our products to work for at least 180 days from the date of purchase when stored correctly.

Storage at +4C for this long of a period may or may not have affected the product. I would recommend use as normal to determine if there has been any adverse affect.

For more information on antibody storage and stability, please visit our Antibody Storage Guide on our Protocols and Troubleshooting Tips page (https://www.abcam.com/index.html?pageconfig=popular_protocols). I have included this as an attached pdf as well.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

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Answer

Thank you for providing that extra information.

The band that can be expected with this lysate is the predicted 517 kDa, however, to our knowledge, this lysate has not been specifically probed for Lrp2 and I cannot therefore be sure of this. Additionally, ab56014 has not been used to detect the endogenously expressed protein in western blotting in house (which is also seems to betrue of the Sc-25470). Only recombinantly expressed protein fragments have been detected. This makes performing the western blot considerably easier as the transfer of the smaller recombinant protein is much less problematic than transferring a 517 kDa protein.

Can I ask, are you seeing any bands after performing the western blot or is the membrane completely blank? If you could possibly share an image of the blot obtained that would be very helpful. What was the result of the "no primary" control?

I would advise tryingthe following in order to improve the resultsobserved to date:

1.I would prepare the protein samples by heating for 10 minutes to 70 degrees. If you are not already doing so I would also used a reducing agent in the loading buffer. This is not necessary with the control lysate as this is already included (butthe heating step should still be performed).

2. In order to improve the transfer and binding of the protein to the membrane I would use PVDF membrane and make sure to pretreat in methanol for 1-2 minutes, followed by incubation in ice cold tranfer buffer for 5 minutes.

3. I would use a very low percentage gel in order to improve the transfer, as well as adding 0.1% SDS to the tranfer buffer as this prevents the precipitation of the protein in the gel. I would also remove the methanol from the transfer buffer as this tends to remove the SDS. Methanol is only necessary in the transfer buffer if using a nitrocellulose membrane. Removing the methanol also promotes the swelling of the membrane which allows large proteins to transfer more easily.

I hope these suggestions improve the results observed so far. Performing a western blot with such a large protein will be quite difficult and may require quite a lot of optimisation in order to get a good result.

I look forward to receiving yourreply.

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Question

1) Abcam product code ab 44687

2) Abcam order reference number or product batch number
GR 316263
3) Description of the problem: no band / high background (many non-specific
bands) / wrong band size / other?
Wrong band size
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell
lysate
Species : Human kidney cells (HK-2)
Lysis buffer : Passive lysis buffer which contains protease inhibitors
Protease inhibitors:
Phosphatase inhibitors :
Reducing agent :
Boiling for ≥5 min? yes (I have boiled my samples, ab 44687 was loaded as
is without boiling)
Protein loaded ug/lane or cells/lane : 40µg
Positive control : human kidney lysate ab 44687
Negative control : HeLa cells
5) Percentage of gel 5%
Type of membrane nitrocellulose
Protein transfer verified Ponceau
Blocking agent and concentration 5% of non fat dry milk in TBST
Blocking time 1Hr
Blocking temperature room temp.
6) Primary antibody (If more than one was used, describe in “additional notes”)
: rabbit anti human megalin
Concentration or dilution 1:200 , 1:500
Diluent buffer blocking solution
Incubation time overnight
Incubation temperature: 4C
7) Secondary antibody: goat anti rabbit HRP
Species: goat
Reacts against: rabbit
Concentration or dilution 1:25,000
Diluent buffer TBST
Incubation time 1hr
Incubation temperature: room remp.
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer TBST
Number of washes 4 times
9)Detection method film exposure
10) How many times have you run this staining? once
Do you obtain the same results every time?
What steps have you altered to try and optimize the use of this antibody?
I've used your kidney lysate with two antibodies: the first one was ab56014
(anti human megalin directed at the N-terminal), which I have tried several
times without getting any bands, and the second was another antibody directed
at the C-terminal (not from abcam). The conditions I've used were fairely the
same with both antibodies, however, I did not get any bands with your antibody.
The new antibody gave me wrong bands (˜200kDa instead of ˜600kDa) even when I
used your human kidney lysate.
I would really appreciate your help in this matter.
Best regards,

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to clarify a few details in order to be able to understand the situation more fully and hopefully be able to provide some suggestions in how to improve the results currently observed.

1. could you share with me what the other anti-Lrp2 antibody used was (manufacturers name and catalogue number)?

2. what lysis buffer was used for your other samples?

3. what transfer buffers were used (composition)?

4. how long was the transfer performed for and at what voltage?

5. you mentioned that the transfer was verified by staining of the membrane, do I assume right that the 500-600 kDa region transferred ok? Could you share the images of theseresults with me?

6. how long was the membraneexposed for?

7. has a "no primary" control been performed?

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for contacting us and reporting the problems observed with using anti-Lrp2antibody ab56014 with Kidney (Human) Tissue Lysate (ab44687).

In order to understand the problems your customer is experiencing further could you please pass on the questionnaire attached to this email for them to fill out. Performing western blotting with such a large protein can be very tricky and fully understanding the protocol your customer has used may shed further light on what may be the problem.


I look forward to receiving your reply.

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