製品の概要

  • 製品名

  • 製品の詳細

    Goat polyclonal to LASP1
  • 由来種

    Goat
  • アプリケーション

    適用あり: WBmore details
  • 種交差性

    交差種: Human
    交差が予測される動物種: Mouse, Rat, Chicken, Pig
  • 免疫原

    Synthetic peptide:

    NPNCARCGKIVYP

    , corresponding to N terminal amino acids 2-14 of Human LASP1.

  • ポジティブ・コントロール

    • WB: Recombinant Human LASP1 protein (ab114888), Mouse brain extracts.
  • 特記事項

    Principal Names: LASP1 (LIM and SH3 protein 1), MLN50. GenBank Accession Number: NP006139 LocusLink ID: 3927 Please note that one of our customers has had problems getting this antibody to detect endogenous Lasp-1 in 2 different human cell lines. Furthermore, a LASP-1/GFP fusion protein overexpressed in a cell line could not be detected with this antibody and yet was detected with our Rabbit Polyclonal to GFP (ab290). For further information please see the enquiries section of the online datasheet. We are currently conducting more QC tests on this product and are interested to hear from other researchers using it. 24 March, 2004.


    This protein functions as an actin-binding protein and possibly in cytoskeletal organization.

製品の特性

  • 製品の状態

    Liquid
  • 保存方法

    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • バッファー

    pH: 7.30
    Preservative: 0.02% Sodium azide
    Constituents: 0.05% Tris, 0.5% BSA
  • Concentration information loading...
  • 精製度

    Immunogen affinity purified
  • 特記事項(精製)

    Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide.
  • 一次抗体 備考

    This protein functions as an actin-binding protein and possibly in cytoskeletal organization.
  • ポリ/モノ

    ポリクローナル
  • アイソタイプ

    IgG
  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab1301 in the following tested applications.

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

アプリケーション Abreviews 特記事項
WB Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 30 kDa (predicted molecular weight: 30 kDa).

ターゲット情報

画像

  • LASP1 staining (0.5µg/ml) of Mouse Brain extracts (RIPA buffer, 35µg total protein per lane). Primary incubated for 1 hour.  Detected by western blot using chemiluminescence LASP1 staining (0.5µg/ml) of Mouse Brain extracts (RIPA buffer, 35µg total protein per lane). Primary incubated for 1 hour. Detected by western blot using chemiluminescence

プロトコール

参考文献

This product has been referenced in:

  • Li K  et al. Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins. J Proteome Res 4:725-33 (2005). Read more (PubMed: 15952719) »
See 1 Publication for this product

レビューと Q&A

1-5 of 5 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (IMR-90 cells)
Loading amount
30 µg
Specification
IMR-90 cells
Gel Running Conditions
Non-reduced Denaturing

Abcam user community

Verified customer

投稿 Apr 22 2009

Answer

Thank you for your email and I'm sorry to hear that you are experiencing some difficulty with these antibodies. All 3 of these antibodies have been successfully tested for application in Western blotting. However, these antibodies have not yet been tested for application in IHC to our knowledge. They very well may not be suitable for this particular application. I do suggest that you submit Abreviews regarding your experiences with these antibodies in IHC as your feedback is extremely useful for us as well as other customers. To submit an Abreview, just click on the Abreviews tab located on the online product datasheet and then click on the submit an Abreview link. In exchange for an Abreview we will award 50 Abpoints (an additional 100 Abpoints if an image is submitted) which can be exchanged for a variety of awards. In order to check to see if your secondary antibody is causing the background that you are seeing, you can test this by omitting primary antibody in your protocol and testing with secondary antibody only. If background appears, change either the secondary antibody or the blocking agent. At the bottom of the online datasheets for these antibodies are a list of compatible secondaries (such as ab6741) which are suitable for use with these primary antibodies. For ab1301 (Goat polyclonal to LASP1), an approximate 30 kDa band was seen in Mouse Brain extracts. I would therefore suggest using this as a positive control. I hope this helps. Please don't hesitate to contact us again if you need additional assistance with these antibodies. Happy New Year!

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Answer

Thank you for your enquiry. I apologize for the confusion, the antibody is antigen affinity purified: Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide. If you have any more questions, please contact us again.

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Question

In response to our conversation on the phone earlier today about the problems with the Goat-anti-Lasp-1 antibody Ab1301, I hereby give you the details about the conditionns that we used to test the antibody. Antibody details: Cat. no.: Ab1301-100 Lot. no.: 21380 First time order Aliquoted at 10 ul and stored at -20 degrees Celcius upon arrival. We used total cell lysates from both HeLa (human cervical carcinoma) and MCF-7 (human mammary adenocarcinoma) cells that were transfected with either GFP alone or GFP-Lasp-1 fusion constructs. These samples were tested in sample preparation buffer containing SDS and beta-mercaptoethanol and were boiled 5 minutes at 95 C. We also split some samples in 2 parts and then one half was not boiled for better antigen conservation. The extracts were separated on a 4-20% gradient SDS-PAGE gel, transferred to nitrocellulose and stained: Blocking: 1 hr at RT rocking in 2.5% or 5% skimmed milk in TBS-T (0.05% Tween-20) Goat-anti-Lasp-1 Ab1301: Overnight at 4 C rocking at 1/500, 1/1000, 1/2500 in 2.5% or 5% milk/TBS-T Wash: 5 minutes at RT rocking; 3-5 x TBS-T Rabbit-anti-Goat-HRP (DAKO): 1 hour at RT rocking at 1/1000 and 1/2000 in 2.5% or 5% milk/TBS-T Wash: 5 minutes at RT rocking; 3-5 x TBS-T ECL: Pierce ECL reagent 1 minute at RT Results: 1/1000 and 1/500 dilutions of Ab1301 in 5% milk yielded some background staining and ~20 bands, the strongest of which were 80 and 50 kDa. There was a very faint band at 30 kDa (native Lasp-1) but it was impossible to say whether this was specific given the high number of bands. In GFP-Lasp-1 overexpressing cells no increase in intensity at 58 kDa could be seen compared to empty vector GFP-overexpressing cells. Blots were stripped and reprobed with Rabbit-anti-GFP Ab290 1/4000 in 1%milk/TBS-T, which resulted in clear GFP (28 kDa) and GFP-Lasp-1 (58kDa) bands. We also tested the secondary antibody alone (without Ab1301) and this resulted in only a few very faint bands at much higher exposure than in the presence of Ab1301. Dilution of Ab1301 to 1/2500 and dilution of secondary Ab to 1/2000 and using 2.5% milk/TBS-T reduced the background staining and the number of bands slightly but the intensity of the 30 kDa band also decreased and still no specific 58 kDa band could be observed in GFP-Lasp-1 overexpressing cells. Using unboiled samples only increased the number of bands with Ab1301. As you can see we have tried a lot of conditions and the antibody still does not work as can be expected since we bought the antibody for detection of endogenous human Lasp-1 and it does not even detect high levels of human Lasp-1 as present in GFP-Lasp-1 overexpressing cells. Therefore, we are asking for a different antibody batch and, if this is not available, for refunding. I am looking forward to hear from you.

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Answer

Your feedback places doubt on the sensitivity/specificity of our antibody and we take it very seriously. As we discussed over the telephone, I will gladly refund you for the cost of this antibody as it clearly has not worked in your research. I'm afraid that we do not have another batch available. The question now is whether this antibody is suitable to stay in our catalog and for us to continue to sell to other researchers. To this end, your comments on the following questions would be appreciated. 1. Please confirm that the construct you used in the transfection does code for the epitope we used to make the antibody (NPNCARCGKIVYP, from N Terminus of the protein sequence according to NP_006139 - sorry but we have to ask this obvious question) 2. Do you think that the prescence of the GFP tag could be interfering with the binding of the antibody (ie is the tag on the N-terminus?)." Thank you again for your feedback and I look forward to your response.

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