Overview

  • Product name

  • Description

    Goat polyclonal to Lhx2/LH2
  • Host species

    Goat
  • Tested applications

    Suitable for: WB, ELISAmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat, Dog, Common marmoset
  • Immunogen

    Synthetic peptide corresponding to Human Lhx2/LH2 (C terminal).
    Sequence:

    C-NLEGHEPHSPSQ


    Database link: NP_004780.3

  • Positive control

    • Daudi cell lysates
  • General notes

     This product was previously labelled as Lhx2

     

Properties

Applications

Our Abpromise guarantee covers the use of ab77368 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
ELISA
  • Application notes
    Peptide ELISA: Antibody detection limit dilution 1/4000.
    WB: Use at a concentration of 1 - 3 µg/ml. Predicted molecular weight: 45 kDa.

    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    Images

    • Anti-Lhx2/LH2 antibody (ab77368) at 1 µg/ml + Daudi cell lysate in RIPA buffer at 35 µg

      Predicted band size: 45 kDa
      Observed band size: 45 kDa

    References

    ab77368 has not yet been referenced specifically in any publications.

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Answer

    Yes, I believe it is worth trying. There is no exact rule on which blocking agent is best for WB. In some cases antibodies do not work with milk but with BSA. The opposite is also sometimes observed. For example, ss shown on the WB image using ab9385, the signal is weak or null using milk. However, with BSA it is very strong and specific. The link to this example is : https://www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg I hope this helps.

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    Question

    Antibody code: ab77368 Batch number: gr9231-1 Antibody storage conditions (temperature/reconstitution etc) Aliquot and -20 Description of the problem (high background, wrong band size, more bands, no band etc.) Only background bands. Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) We tried this experiment a few times, using: 1. Mouse embryos (E9.5), in which Lhx2 is highly expressed, 2. Mammalian cell line (C2C12) in which we over-expressed Lhx2 and confirmed it with Real-time PCR. Sample preparation (Buffer/Protease inhibitors/Heating sample etc.). We tried to buffers, either RIPA or triton based lyses buffers. We used the P8340 protease inhibitors by Sigma and heating was for 5’ at 95C.  Amount of protein loaded: 50ug Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) : We did 12% gel SDS reducing gel. We also tried non-reducing (without SDS and DTT). Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) : Transfer was done O/N with MeOH buffer and PVDF membranes. For blocking we used 5% milk in TBST. Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) : We used it according to the rage recommended in Abcam datasheet. We incubated the antibody either in RT for 1H or O/N at 4C.  3 washes in TBST were applied, 10’ each. Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) : We used donkey-anti Goat HRP by Jackson. 3 washes in TBST were applied, 20’ each Detection method (ECL, ECLPlus etc.) For detection, we used ECL kit by Amersham. Positive and negative controls used (please specify) : As mentioned above, Mouse embryos (E9.5), in which Lhx2 is highly expressed, were used as positive controls. As negative controls, we used Lhx2-null embryos. Optimization attempts (problem solving) How many times have you tried the Western?  6 Have you run a "No Primary" control? We did, and we did not get any ‘non-specific’ bands. Do you obtain the same results every time? e.g. are the background bands always in the same place? Yes they are. What steps have you altered? I have noted above, in each step, all the possibilities I have altered during the experiment. Additional Notes : I have previously used many antibodies by Abcam, both for IHC and WB. Besides the case of the last two Lhx2 Abs, I was always happy with the results.

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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 offer some suggestions to help optimize the results from ab77368 : - When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : Click here for the western blot image using ab9385 (or use the following: www.abcam.com/ab9385). Should the suggestions not improve the results, please do let me know. To our knowledge, this antibody has not been used yet on Mouse samples, however, the homology between the immunogen and the Mouse protein is 100%, so I am happy to inform you that the Abpromise guarantee can be applied on this occasion. Therefore we would be pleased to provide a free of charge replacement, or a credit note. I hope this information is helpful, and I thank you for your cooperation.

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