Overview

  • Product name
    NIH 3T3 whole cell lysate
    See all NIH 3T3 lysates
  • General notes
    Cell line: NIH/3T3 (Mouse embryonic fibroblast).
    Growth media: DMEM and 10% bovine calf serum.


    Mouse NIH/3T3 cell lysate was prepared by homogenization in modified RIPA buffer(50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% sodium dodecylsulfate (SDS), 1 mM sodium ethylenediaminetetraacetate,1 mM phenylmethyl-sulfonyl flouride, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin). Cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% SDS, 0.01% bromophenol blue) containing 5% b-mercaptoethanol.

  • Tested applications
    Suitable for: WBmore details

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot. Store at -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Constituent: 5% Beta mercaptoethanol
  • Concentration information loading...
  • Purity
    Whole Cell Lysate
  • Lysate notes
    Mouse NIH/3T3 cell lysate was prepared by homogenization in modified RIPA buffer(50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% sodium dodecylsulfate (SDS), 1 mM sodium ethylenediaminetetraacetate,1 mM phenylmethyl-sulfonyl flouride, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin). Cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% SDS, 0.01% bromophenol blue) containing 5% b-mercaptoethanol.
  • Research areas
  • Background
    NIH 3T3 cells are established from a NIH Swiss mouse embryo. These cells are highly contact inhibited and are sensitive to sarcoma virus focus formation and leukaemia virus propagation. Cells have now lost their contact inhibition. This cell line was established from NIH Swiss mouse embryo cultures in the same manner as the original random bred 3T3 and the inbred BALB/c 3T3. The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays. It is therefore used for DNA transfection studies

Applications

Our Abpromise guarantee covers the use of ab7179 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.
NIH/3T3 cell lysate is ready to load on SDS-PAGE for Western blotting, 20 µg per lane is recommended for mini gel.

References

ab7179 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-5 of 5 Q&A

Answer

Thank you for contacting us.

Based on this image alone it seems like the lower band is your CDKN2A/p19ARF band. It is possible for band size to change slightly due to sample preparation or buffer, however usually the positive control will be a good indicator of where the protein of interest is. One strategy to confirm the band might be to use the blocking peptide (the peptide used as the immunogen) to specifically block the CDKN2A/p19ARF protein. We have this available as ab
2659.


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

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Answer

Thank you very much for contacting us with your enquiry.

I'm sorry to hear that there were no bands in Western blot using this antibody. I would expect any of the MEF cell lines to work as a positive control, but I suggest using a whole cell lysate (ab14874 is a nuclear fraction) since that is what we have tested with ab80. What kind of samples are you using other than the MEF lysate?

If you'd like to send some more details about your protocol ( how much protein was loaded, dilution of antibody, blocking solution, etc) I'd be happy to take a look and see if we have any suggestions to improve the results.

I look forward to hearing from you. If there's anything else that we can do for you, please let me know and I'll be happy to help.

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Answer

I apologize for the delay in getting back to you. I am still waiting for a reply from our source of the skeletal muscle lysate. I do not think it should contain alpha-SMA.

On the other hand, the fault may be with the antibody, if is not detecting this protein in the NIH3T3 lysate. I suspect that the antibody, though we have not received other complaints about it recently, is cross-reacting with something in the skeletal lysate that happens to be the same size as SMA.

Have you tried staining blots of these lysates with any other antibodies? If so, were the results as expected?

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Answer

Thank you for contacting Abcam and for your interest in our whole cell lysates.

Regarding these products, they are not tested for protein composition. They are only tested for protein concentration and confirmed to not be degraded.

If you are interested in determining which lysates will contain the protein that you need, I would recommend a search of the literature. Unfortunately, we are not experts in this field and would not want to recommend a product that is not suitable for your experiments.

I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

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Answer

Thank you for your enquiry. You are correct, this antibody is at a concentration of 2mgml-1. The antibody is provided at 100 µg therefore 50ul. We do receive some very occasional shortages which requires our lab team taking a vial and adding lysate to the other vials that are short of antibody. This can result in customers receiving more antibody or lysate than expected. I would recommend that you prepare 20ug (20ul) of lysate. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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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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