Mouse lung normal tissue lysate - total protein (ab29297)

Q&A (4)

Overview

  • Product name
    Mouse lung normal tissue lysate - total protein
    See all Lung lysates
  • General notes


    Protein is isolated from whole tissue homogenates using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70ºC. Quality control: 1. The isolated protein pattern on SDS-PAGE gel is visualized by coomassie blue staining. The pattern is consistent with each lot. 2. The isolated protein is Western Blot analyzed by either a GAPDH or beta-actin antibody. The expression level is consistent with each lot. Ethics: Human tissue samples are collected with the informed consent of the donors & relatives and are excised by licensed medical doctors. Tumor tissue samples are diagnosed and identified by at least two different evaluators. If you have more questions then please feel free to contact us. Donor information can be supplied on request.

  • Tested applications
    Suitable for: WBmore details

Properties

  • Form
    Liquid
  • Storage instructions
    Store at -80°C. Allow to warm to room temp and agitate gently before aliquotting.
  • Storage buffer
    Constituents: 1% Tergitol-NP40, Potassium chloride, 0.1% Bromophenol blue, Magnesium chloride, 1.2% Beta mercaptoethanol, 2% SDS, HEPES, Sucrose, 10% Glycerol

    With Protease inhibitor cocktail/EDTA/Sodium deoxycholate
  • Concentration information loading...
  • Lysate notes
    Protein is isolated from whole tissue homogenates using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70ºC. Quality control: 1. The isolated protein pattern on SDS-PAGE gel is visualized by coomassie blue staining. The pattern is consistent with each lot. 2. The isolated protein is Western Blot analyzed by either a GAPDH or beta-actin antibody. The expression level is consistent with each lot. Ethics: Human tissue samples are collected with the informed consent of the donors & relatives and are excised by licensed medical doctors. Tumor tissue samples are diagnosed and identified by at least two different evaluators. If you have more questions then please feel free to contact us. Donor information can be supplied on request.
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab29297 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 dilution. Samples need to boiled for 5 minutes and put on ice before use.

References

ab29297 has not yet been referenced specifically in any publications.

Publishing research using ab29297? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

Q&A
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1-4 of 4 Q&A

Question

would it be possible for us to get another vial of the ab81673 antibody as we talked about on Thursday? Our original purchase order number for the ab42885 was 1263787. We are going to try to immunoprecipitate VAP-1 from the lysate then detect it via western blot. Let me know if you think this would work, or if you have any other suggestions. e-mail.

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Answer

Thanks for your reply.

ab81673 has been tested for use in WB and IP so the experiment you propose should be fine. An alternative is to use one VAP1 antibody for IP and second VAP1 antibody for WB.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab48281.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question

I'm sorry if this wasn't clear in the last email, but we actually used up the lysate sent to us when repeating the experiment trying to get it to work, so it would be impossible to repeat these tests without a new lysate. Our initial question was to find out if the 75 kDa band we see in our lysate is in fact VAP-1. You have reported on the website that the antibody ab42885 detects VAP-1 as an 85-100 kDa band in lung lysate. You sent us your lung lysate as a positive control, and we were not able to detect VAP-1. We need that control in order to interpret our studies. Can you help us with this?

Also, the antibody we've been using for FACS, ab81673, has not been reported or guaranteed to work in western blot. We bought the second antibody, (ab42885) specifically because it was guaranteed to detect VAP-1 in a western blot. If we can't get it to work, or if you all don't have a different suggestion for changing the protocol, would it be possible for us to get a credit for this antibody, or a second ab81673 for free? I know you've sent us some free products, which again I really appreciate, but at the end of the day, if we can't get them to work at all when we bought them for one specific purpose, I feel we should get a credit. I really hope you can work with us by giving us scientific support with this problem.

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Answer

Thanks for your reply.

Perhaps it would be more efficient to discuss this issue on the phone. Please feel free to call me. Alternatively, if you provide a phone number I'm happy to give you a call.

I look forward to speaking with you. I'm sure we will quickly come to an agreeable solution.

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Question


I attached our previous run of the lysate you sent us next to one we had. The one we’ve been using still produces a 75 kDa band, rather than the 85-100 kDa band that your website lists as VAP-1. As you can see, the lysate you sent us produces a much lower band under the same conditions.

It’s possible that this 75 kDa band is VAP-1, and for whatever reason we’re getting a slightly lower band than we should. We would believe this is the case if we could corroborate it with a positive control, but the lysate you sent us didn’t work as planned, either with the original antibody we bought or with the new antibody you gave us. It produced a much lower, 37 kDa or so band.

Abcam has sent us a free antibody and a free lysate, which we really appreciate, but they just have not worked in our hands the way your website guarantees they should. I’m not sure if this is a problem with the antibody itself, the lysate we had, or with our conditions, but it isn’t working the way we need it to. We think the lysate you gave us may be the issue because its results are very different from anything else we've run with either antibody. We’re currently using reducing conditions, so if there are other suggestions you may have that might help, that would be great.

The other antibody we have for VAP-1 is ab81673, which we haven’t used in western blotting because it isn’t guaranteed for western blot. This one does work fine for us for other uses.

We’d really like a different lysate to use as a positive control, because our other loading controls don’t produce a band in your recommended range.

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Answer

Thanks for your email.

Since you have ab81673 I would recommend using it in a western blot with the 2 lysates you have been testing. It is quite likely this antibody will work in western blotting as it has already been shown to work in a diverse application range.

Thanks for all your troubleshooting efforts. I look forward to hearing how this next western blot goes.

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Question


It's been a little while since I emailed you, so to refresh your memory I'm Andrew Knapp from Dr. Pilar Alcaide's lab. We're moving back toward VAP-1 western blotting experiments, and we're again having issues with finding VAP-1 in lysate tissues. I wanted to write you with the problems we've been having, and see if there were any solutions or ideas you might have.


1) When running our samples against the lysate you sent us as a control, our samples come out around 75 kDa and the lysate has a band around 37 kDa. Neither of these are in the 85-100 kDa range that your website suggests for VAP-1.

2) When running immunoprecipitate samples, we see a band around 150 kDa. Have you ever done this or seen this result?

3) Do you typically recommend running in reduced or non-reduced sample buffer? We have been running them in reduced sample buffer, and were not sure if that makes a difference.

4) Would it be possible for us to have another lysate from you to use as a control? You originally sent us a lung cell lysate (ab29297) to try. Could we have another of these, or is there a different lysate you have been able to detect VAP-1 in that might work for us?

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Answer

Thanks for your email.

The data you are seeing do appear unexpected.

Reducing conditions are used during western blotting.

Before I believe a loading control western blot was producing expected banding pattern so I don't think this is an issue with the lysates. More likely it appears to be an issue with the antibody.

Have you used any other anti-VAP-1/AOC3 antibodies to test for reactivity via western blotting?

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