Overview

  • Product name
    HepG2 whole cell lysate
    See all HepG2 lysates
  • General notes
    Cell line: HepG2 (Human hepatocellular carcinoma).
    Growth media: DMEM and 10% FBS (Fetal bovine serum).


    HepG2 cell lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl flouride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 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 cell lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 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
    HepG2 cell lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl flouride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 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 cell lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol.
  • Research areas
  • Background
    Hepatitis B virus (HBV) is the major cause of acute and chronic hepatitis, leading to progressive development of necroinflammatory changes in the liver, which can result in cirrhosis and hepatocellular carcinoma. Although the development of an effective vaccine to prevent HBV infection has shown promising results and should lead to its eventual eradication, antiviral chemotherapy remains the only effective method to prevent the progression of the disease in chronic carriers. Therefore, the development of new antiretroviral agents active against HBV is needed. HepG2 cells have an epithelial morphology and are thought to be a very useful model to study HBV virus replication via transfection. Cells are also used for cancer and apoptosis studies (in particular signalling pathway studies).

Applications

Our Abpromise guarantee covers the use of ab7900 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 concentration. HepG2 cell lysate is ready to load on SDS-PAGE for Western blotting, 20 µg per lane is recommended for mini gel.

References

ab7900 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-10 of 10 Q&A

Answer

Thank you for contacting us.
The exact protocol is proprietary for ab14659, but the cytoplasmic membrane is first lysed in the hypotonic buffer to harverst the nuclei. Then the proteins from the nuclei are extracted with a lysis buffer. The samples are not boiled.
Are of our lysates have been treated with detergents.
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 for your technical support and interest in our products.

As the datasheet indicates, the recommended positive control for ab89220: Anti-Tbx3 antibody is HepG2 cell lysate for Western blot application.

I have conducted a search for you and found 8 HepG2 cell lysate in our catalogue. You can use whole cell lyaste such as ab7900 or nuclear lysate (untreated) such as ab14660. Since Tbx3 is localised in the nuclear, it may be worth using the nucler fraction and enriched lysate.

I would advise you to take a look at these products to see which one would suit your need best:

https://www.abcam.com/ab7900 HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

https://www.abcam.com/ab14660: HepG2 (Human hepatocellular liver carcinoma cell line) Nuclear Lysate

If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for contacting Abcam.

I believe we spoke on the telephone earlier regarding this question.

As I mentioned the modified RIP buffer was used as it was found to give slightly more stable results compared to regular RIPA buffer. The modified buffer is not available from our catalogue but the recipe is available online and so would be very simple to make in your lab. You could just use regular RIPA buffer and manually adjust the modified components.

As to whether you would get the same results or not, I cannot guarantee they would be identical, but they should be comparable.

I am sorry I could not help further in this case, but if you have any other questions please let me know.

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Answer

Thank you for your inquiry.

Catalog number ab7900 is a whole HepG2 cell lysate that is provided in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol. It is at 2 mg/ml.
If you are interested in a bulk quote, please contact my colleagues Andrea and Jimmy at mailto:sales@abcam.com.
I hope this information helps. Please contact us with any other questions.

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Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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 for one vial of ab7900replacement with the order number 1148322.

Expect to receive this product on Tuesday August 21. 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
Answer

Thank you for contacting us.

As a positive control I would recommend using a HepG2 lysate such as ab7900. For a negative control, this same lysate can be de-phosphorylated using this protocol:

https://www.abcam.com/index.html?pageconfig=resource&rid=11407

I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for contacting Abcam.

I am sorry about the issues you have been having with ab108447 in western blot. The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on human. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

I just had two quick protocol questions about the HepG2 samples that you were using:

1 - Did you try loading 20ul of sample? As that is how much we recommend to load.

2 - Before loading the lysate, did you boil the samples?

I look forward to your reply and helping resolve this issue.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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 with the order number 1075950.

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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Answer

Thank you for contacting us.

After further investigation, I found that the WB application was added by error which was removed a week after. The reason for removal was, non availability of tested data to prove the suitability. However this does not mean that the antibody cannot be used in WB it only mean we can't support this as tested application. I apologize for any inconvenience this may have caused.

I would suggest troubleshooting the protocol as per recommendations given in my previous email or I can also send you free of charge replacement antibody. We have ab126208 in stock which was tested in WB and is fully guaranteed. Could you let me know how you would like to proceed.

I once again apologize for error. I will look forward to hearing from you soon.

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Answer

Thank you for your email. I am sorry to hear that you have been experiencing difficulties using this antibody.

We have not tested this antibody before in western blot so we are not sure at what molecular weight the band will be observed. This antibody was never published as applicable in western blot. Could you send me the datasheet with wb application on it. Unfortunately because this antibody was never tested in WB so this product is not covered under Abpromise guarantee, we will not be able to offer you refund or replacements.

I however can help you with troubleshooting

- GPCR is a multipass membrane protein which forms aggregate when heated at 100C in western blot. We recommend heating the sample for 15-20 minutes at 70-80C.
- Please make sure the lysis buffer contains protease inhibitors and it is ice cold.
- Please try using NP40 buffer for cell lysis
- The confluency before lysis should be 90%

Please note that the observed band size will not be 88kDa, I will be slightly less because the first 26 amino acids are cleaved on while post translational processing. Also this protein is very heavily glycosylated the observed band size will be higher in molecular weight then predicted 88kDa.

I have few further questions
- Why there is no band in third lane MCL3.

I hope these suggestions will help to improve your results. Please do not hesitate to contact me if you have any question.

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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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