Overview

  • Product name
  • Description
    Rabbit polyclonal to LAP2 alpha
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IP, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment (His-tag) corresponding to Human LAP2 alpha aa 188-693.

  • General notes


    Lamins are type V intermediate filament proteins and are grouped into constitutively expressed B-type lamins and developmentally regulated A- type lamins. Lamin-binding proteins in the nuclear lamina and the nuclear interior include several protein families and/or types of proteins in higher eu karyotes such as the inner nuclear membrane proteins, lamin B receptor, emerin, and MANI, three isoforms of lamina-associated polypeptide 1 (LAP 1), and several isoforms of LAP 2. Up to six LAP 2 isoforms derive from a single gene by alternative splicing in mammals and various isoforms have been described in Xenopus. The best characterized LAP2 isoforms are the inner nuclear membrane protein LAP 2 beta and the nucleoplasmic protein LAP 2 alpha, which are identical in their N-terminal 187-amino acid constant region but differ in their C termini. While LAP 2 beta binds to B-type lamins at the nuclear periphery and was suggested to regulate nuclear lamina growth , LAP 2 alpha specifically interacts with A-type lamins within the nuclear interior as part of a detergent/salt-resistant nucleoskeletal structure.

Properties

  • Form
    Liquid
  • Storage instructions
    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.
  • Storage buffer
    Preservative: 0.1% Sodium azide
    Constituents: PBS, Whole serum
  • Purity
    Whole antiserum
  • Primary antibody notes
    Lamins are type V intermediate filament proteins and are grouped into constitutively expressed B-type lamins and developmentally regulated A- type lamins. Lamin-binding proteins in the nuclear lamina and the nuclear interior include several protein families and/or types of proteins in higher eu karyotes such as the inner nuclear membrane proteins, lamin B receptor, emerin, and MANI, three isoforms of lamina-associated polypeptide 1 (LAP 1), and several isoforms of LAP 2. Up to six LAP 2 isoforms derive from a single gene by alternative splicing in mammals and various isoforms have been described in Xenopus. The best characterized LAP2 isoforms are the inner nuclear membrane protein LAP 2 beta and the nucleoplasmic protein LAP 2 alpha, which are identical in their N-terminal 187-amino acid constant region but differ in their C termini. While LAP 2 beta binds to B-type lamins at the nuclear periphery and was suggested to regulate nuclear lamina growth , LAP 2 alpha specifically interacts with A-type lamins within the nuclear interior as part of a detergent/salt-resistant nucleoskeletal structure.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab5162 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
IP Use at an assay dependent dilution.
ICC/IF Use at an assay dependent dilution.
WB 1/2500. Detects a band of approximately 83 kDa.

Target

  • Relevance
    Lamins are type V intermediate filament proteins and are grouped into constitutively expressed B-type lamins and developmentally regulated A-type lamins. Lamin-binding proteins in the nuclear lamina and the nuclear interior include several protein families and/or types of proteins in higher eukaryotes such as the inner nuclear membrane proteins, lamin B receptor, emerin, MANI, three isoforms of lamina-associated polypeptide 1 (LAP 1), and several isoforms of LAP 2. Up to six LAP 2 isoforms derive from a single gene by alternative splicing in mammals and various isoforms have been described in Xenopus. The best characterized LAP2 isoforms are the inner nuclear membrane protein LAP 2 beta and the nucleoplasmic protein LAP 2 alpha, which are identical in their N-terminal 187-amino acid constant region but differ in their C termini. LAP 2 alpha specifically interacts with A-type lamins within the nuclear interior as part of a detergent- and salt-resistant nucleoskeletal structure.
  • Cellular localization
    Nuclear
  • Database links
  • Alternative names
    • Isoforms beta/gamma antibody
    • Lamina associated polypeptide 2 antibody
    • LAP2 antibody
    • Thymopoietin antibody
    • Thymopoietin isoform alpha antibody
    • TMPO antibody
    • TP alpha antibody
    see all

Images

  • ab5162 at a dilution of 1/250 staining LAP 2 alpha in HeLa cells by immunofluorescence.

    A = ab5162, 1/250

    B = DNA

    C = A and B merged

  • ab5162 at dilutions of 1/100 and 1/500 staining approximately 83 kDa LAP 2 alpha in HeLa cell extracts by Western blot (ECL). ab5162 at dilutions of 1/100 and 1/500 staining approximately 83 kDa LAP 2 alpha in HeLa cell extracts by Western blot (ECL).
  • LAP2 alpha was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to LAP2 alpha (ab5162) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab5162.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 83kDa: LAP2 alpha.

References

This product has been referenced in:
  • Khanna R  et al. E3 ubiquitin ligase RNF123 targets lamin B1 and lamin-binding proteins. FEBS J 285:2243-2262 (2018). Read more (PubMed: 29676528) »
  • Bártová E  et al. Depletion of A-type lamins and Lap2a reduces 53BP1 accumulation at UV-induced DNA lesions and Lap2a protein is responsible for compactness of irradiated chromatin. J Cell Biochem N/A:N/A (2018). Read more (PubMed: 29923310) »
See all 11 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (U2OS)
Specification
U2OS
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Oct 22 2014

Answer

Thank you for your enquiry.

I can confirm that ab5162(Anti-LAP2 alpha antibody) is sold as whole antiserum. Unpurified antibodies, such as those sold as whole antiserum, ascites or tissue culture supernatant will not have a concentration stated on the datasheet.

Antibody concentration is usually determined by protein assay, and serum will contain a lot of other proteins, which means the antibody quantification would not be accurate.

I can confirm that for whole antiserum, concentration of antibody is known to vary between 1 - 5 mg/ml.

I am sorry we are not able to provide an exact concentration on this occasion, but hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Question
Answer

Thank you for kindly confirming these details. I am sorry this vial of antibody has not worked for this customer.

As requested, we are pleased to arrange a free of charge replacement. I can confirm that 1 vial of ab5162 has been added to your order number #####. This has Abcam order reference ######.

I would like to reassure you that this free of charge replacement vial is also covered by our Abpromise guarantee. Should the customer still be experiencing difficulties with the new vial, or if you have any further questions, please do not hesitate to let me know.

Thank you for your help and cooperation with this case. Please do not hesitate to contact me if you need anything further.

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Question
Answer

Thank you for your reply.

I am sorry to confirm that we don't have any different lots in stock at the moment. I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Regrettably, I can suggest you have received a bad vial on this occasion.

Therefore, we would be pleased to provide a replacement from the same lot, which will still be covered by our guarantee. Alternatively, I fully understand the customers concerns and you prefer a credit note in this case, I will be pleased to arrange this for you.

I hope this will be helpful. I look forward to hearing from you with details of how you would like to proceed.

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Answer

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.

I am sorry that because of the number of products we have on the catalog, it is not possible for us to store sample vials to send to customers. I can reassure you that any replacement vial would also be covered by the 6 month guarantee.

I look forward to hearing from you with details of how you would like to proceed.

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Question

Product code: 5162
Lot number: GR17174-4
Inquiry: Please read below the details of customer's complaint:
Antibody code: ab5162
Batch number: Lot GR 17174-4
General Information:
Antibody storage conditions (temperature/reconstitution etc) -20C

Description of the problem: strong background, also on the cells with Lap2Alpha depletion (Lap2Alpha-/-)
Sample (Species/Tissue/Cell Type/Cell Line etc.) MEF cells

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.) Paraformaldehyde

Permeabilization step 3 min with 0.5 %Triton

Blocking conditions (Buffer/time period, Blocking agent etc.) 5%BSA for 20min

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Invitrogen Alexa Fluor 488 goat antirabbit (H+L), 1:1400, incubation 60min, 3 times washing*5min

Detection method Confocal microscope

Positive and negative controls used (please specify) cells with Lap2Alpha-/- and Lap2Alpha+/+ were used. In the cells with Lap2Alpha depletion there were no strong staining in the nucleus, but t non specific signal was detected from the cytoplasm. Secondary antibody was used in the same experiment for LaminA-/- and LaminA +/+ and it gave perfect results.

Optimization attempts (problem solving): different delitopns of Lap2 alpha antibody (did not help)

How many times have you tried the ICC? 2times, ech time the same result, perfect for LaminA antibody and with high background for Lap2lpha antibody.

Have you run a "No Primary" control? not

Do you obtain the same results every time? yes

What steps have you altered? Additional Notes We bought 2 antibody and run the experiment simultaneously with both of them LaminA antibody gave fantastic results, while Lap2Alpha was not specific, even in the cells with Lap2Alpha depletion.

Thanks in advance for your advise.
Regards,

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab5162 is tested and covered by our 6 month guarantee for use in ICC-IF and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Although the other antibody has worked well using this procedure, individual antibodies will often require optimization. I would like to investigate this further and hopefully offer some suggestions to help optimise the results from ab5162. I would also appreciate if you can confirm some details of the procedure:

1. Please provide the order number and date of purchase. Was this Po 88783 ordered 12th Feb?

2. I would appreciate if you are able to provide further details of the primary antibody. What dilution was used, has this been optimized? How long was it incubated for and at what temperature?

3. I would appreciate if you are able to provide an image which would help us to assess the results.

Thank you for your cooperation. I look forward to hearing from you with the further requested details.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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