HEK-293 whole cell lysate (ab7902)

Submit a review Q&A (9)References (1)

製品の概要

  • 製品名

    HEK-293 whole cell lysate
    HEK293 ライゼート 製品一覧

  • 製品の詳細

    HEK-293 lysate

  • 特記事項

    Cell line: HEK293 (Human embryonic kidney cell line).
    Growth media: RPMI and 10% FBS (Fetal bovine serum).


    293 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 minutes 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.

  • アプリケーション

    適用あり: WBmore details

製品の特性

  • Mycoplasma free

    Yes

  • 製品の状態

    Liquid

  • 保存方法

    Shipped at 4°C. Upon delivery aliquot. Store at -80°C. Avoid freeze / thaw cycle.

  • バッファー

    pH: 7.2
    Constituent: 5% Beta mercaptoethanol
  • Concentration information loading...

  • ライゼート 備考

    293 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 minutes 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.

  • 研究分野

  • 背景

    Human Embryonic Kidney (HEK) 293 cells were originally derived from human embryonic kidney and subsequently demonstrated to be a useful cell type to produce adenovirus, other viral vectors, and effectively glycosylated human recombinant proteins. They are therefore used for virology studies and transfected to express recombinant proteins for studies in a number of research fields.

アプリケーション

The Abpromise guarantee

Abpromise保証は、 次のテスト済みアプリケーションにおけるab7902の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。

アプリケーション Abreviews 特記事項
WB
Use at an assay dependent dilution.
293 cell lysate is ready to load on SDS-PAGE for Western blotting, 20 µg per lane is recommended for mini gel.
特記事項
WB
Use at an assay dependent dilution.

293 cell lysate is ready to load on SDS-PAGE for Western blotting, 20 µg per lane is recommended for mini gel.

プロトコール

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

参考文献 (1)

ab7902 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab7902 は 1 報の論文で使用されています。

  • Ferchaud-Roucher V  et al. Maternal obesity results in decreased syncytiotrophoblast synthesis of palmitoleic acid, a fatty acid with anti-inflammatory and insulin-sensitizing properties. FASEB J 33:6643-6654 (2019). PubMed: 30811959

レビューと Q&A

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1-9 of 9 Abreviews or Q&A

Question

Do you supply the positive control to go with ab5231 anti-S6K1 (phospho T229)?

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Answer

We do supply the positive control for the Anti-S6K1 (phospho T229) antibody (ab5231).


The positive control is HEK293 (Human embryonic kidney cell line) Whole Cell Lysate (ab7902). Please refer to the Cell Lysate datasheet for further information.


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Question

Product code: 7902
Inquiry: Dear Sir/madame I have already purchased “Anti Glut2 ab95256 antibody”. For a positive control in WB would it be a good option to purchase the “HEK293 Human embryonic kidney cell line Whole Cell Lysate” instead of liver or pancreas tissue lysate as a positive control for my western blots? Sincerely

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Answer

Thank you for contacting us.

HEK293 cells can also be used as positive control.

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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Question

Hi I just wanted to make sure that we are being credited for these products. Please let me know if our account will be credited or if you need any additional information. Thanks! Amy

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Answer

Thank you for contacting us. I am sorry for the confusion. I was unsure whether you wanted replacement product or a credit note/refund. I have issued credit notes/refunds for these products. Your credit note ID for AB93922 is xxx. Your credit note ID for AB7902 is xxxxx. I am sorry that this antibody did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department. The credit note ID is for your reference only and does not automatically guarantee the credit. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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Question

The antibody and lysate were both purchased under PO# xxxx. As for the 2 new antibodies you suggested, can I get some of those under the program in which someone tests your antibodies in new applications? Also, do you have any information as to what cell lysate or other positive control I could use? Any information on other Western blot conditions? Lysate preparation? or better yet a lysate that you have so kindly made for me? Should I just use anti rabbit HRP with ECL? or would you suggest Biotin streptavidin amplification? FACS conditions? Any information would be greatly appreciated!!

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Answer

Thank you for sending this information.

If you would like, I can issue free of charge replacements of the antibody and lysate with another anti-ThPok antibody such as ab23388 or ab70991 and our recommended positive control lysate. Like ab93922, these antibodies will be covered by our Abpromise guarantee for use in WB on human tissues.

As neither antibody has been tested in FACS, I would be glad to also enroll either of these products in our AbTrial program for that application. Here is a brief description of how this program works:

The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price (in this case I would be providing a product as a free of charge replacement), test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here:https://www.abcam.com/AbTrial

We feel that an HRP- ECL detection system will generally be adequate for use with our WB tested products unless stated on the datasheet. We do not have specific FACS recommendations as we have not yet tested these in that application. I can recommend a starting concentration of 1ug/ml; however the optimal concentration will have to be found experimentally. Our direct and indirect FACS protocols may be found at the following locations:

Direct:https://www.abcam.com/index.html?pageconfig=resource&rid=11380

Indirect:https://www.abcam.com/index.html?pageconfig=resource&rid=11381

More protocol information may be found at:

https://www.abcam.com/index.html?pageconfig=popular_protocols

I hope this information is helpful to you and look forward to hearing how you would like to proceed. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question

So, if you remember I had used your HEK293 whole cell lysate cat ab7902 as a positive control for your anti ThPOK antibody #ab93922 in a Western blot but hadn't gotten any bands. You asked me to try again using a new HRP antibody as the one I had used was from 2009. So, I retested using 10 µl=20 µg of the whole cell lysate and using the 7C12 at 1/500 in 5% skim milk with an HRP secondary from Jackson that I reconstituted on the day of the Western (Goat anti Mouse IgG HRP cat #715-035-151) at the highest suggested conc for westerns of 1/10,000. However, despite good transfer as shown by the MW marker, I didn't see any bandsexcept possibly at >190 kDa with the lysates I made myself for PBMC and 293F in lanes 3&4 respectively. See attached film of blot -left hand = light blot- with an ~2 minute exposure. The 4 - 20% gel was loaded as follows: 1) MW marker 2) HEK293 whole cell lysate 3) PBMC lysate 4) 293F lysate 5) MW marker 6) EL4 lysate The overexposed blot on the right hand side is when I blocked with 2%BSA in TBST, used your antibody diluted in block 1 to 500, used biotin anti mouse Jackson Cat# 115-066-146, and used SA-HRP and ECL. This blot comes from a transfer of the other half of the gel used for the blot above loaded as follows: 7) MW marker 8) HEK293 whole cell lysate 9) MW marker 10) PBMC lysate 11) 293F lysate 12) EL4 lysate If you refer to the "light exposure" attachment you'll see that even with the biotin- SA amplification I didn't get any bands for your lysate. When I used our fancy imager on the blots I saw some bands for your lysate between 60kDa and 85 kDa but that is only with Biotin streptavidin and multiple bands are seen for all lanes including the EL4 lane (which is a mouse cell line so presumably the mouse Ab wouldn't react to mouse proteins), and as you see aren't very convincing. Therefore, I would appreciate being credited for both the antibody and the cell lysate since they didn't perform as expected. More importantly, I would appreciate any ideas that you have that would help me to find an anti human ThPOK antibody that would work for Western and, if I get to have my fantasies fulfilled, FACS!

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Answer

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments, and would like to offer refund or credit note in compensation (providing the product has been purchased in the last 180 days of contacting us). In order to arrange this, I would appreciate if you could confirm your order numbers or PO and dates of purchase for these antibodies and lysate?

We believe that any product which has been tested for ThPOK in WB should perform as stated in the datasheet which is why we offer our Abpromise guarantee. We do have other products which are tested in WB but unfortunately none have also been tested in FACS. When choosing a product which may have the best chance to work in both applications I would suggest using a product which has been tested in ICC, IHC or IP as well as in Western blot. Two products which may perform best in both WB and FACS may be:

AB70991https://www.abcam.com/ThPok-antibody-ab70991.html

AB23388https://www.abcam.com/ThPok-antibody-ab23388.html

Thank you for your continued cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Question

Customer has kindly contacted us regarding ab93922 as it gave no signal in WB when using the recommended HEK293 control.

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Answer

Thank you for your telephone inquiry this afternoon. I am sorry to hear you have experienced difficulty obtaining satisfactory results from this antibody in western blotting.

I do hope the use of a new secondary antibody will resolve this issue for you. However should these issues continue please know that this product is covered by our Abpromise guarantee. We are happy provide scientific support at any time. If you are using the product in species and applications listed on the datasheet and contact us within six months of purchase, we are also happy to replace or refund the product should we not be able to help you to resolve the issue. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Question

Do you guys have a positive control for Anti-Cdk2 antibody (ab6538)? I would like to purchase this antibody with a tested positive control. Thanks! sincerely,  

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Answer

Thank you for contacting us. I presume you are looking for positive control used in western blot. Our customers have used this ab successfully with mouse brain tissue and 293 cells lysates. Please check the Abreviews. The Mouse brain lysates can be found with catalogue numbers are; ab7189, ab7190 ab30151 and Human 293 cell lysates with catalogue number ab7902. Please also click the following link to see the tissues which express CDK2 target, lysates of these can be used as positive control. http://www.proteinatlas.org/ENSG00000123374/normal http://www.proteinatlas.org/ENSG00000123374/cancer http://www.proteinatlas.org/ENSG00000123374/cell We have the tissue lysates in catalogue. Please type "lysates" in the product search box provide on the Abcam homepage and system will give you sorted products as per your requirements. 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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Question

do you sell the lysate from HEK293 cells overexpressing human FOXP3, as used as a positive control for your foxp3 antibody ab22510? If not, would you be able to supply me with a small quantity of cells/lysate? I have been unsuccessful in detecting Foxp3 in human regulatory T cells using ab22510 and a postive control lysate would determine whether my concerns over the antibody are justified. My loading control antibody is working fine so its just the Foxp3 Im having problems with.

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. Unfortunately we do not sell the positive control employed as a positive control for ab22510; HEK293 cells overexpressing human FOXP3. However, we do sell Tonsil (Human) Cytoplasmic Lysate - tumor tissue (ab29606) and tonsil is known to express high levels of FOXP3. It would help me if you could provide me with further information of the protocol that you have been using by clicking on the link below and filling in our technical questionaire. This will better enable our technical team to determine the steps that you have taken to optimise this antibody. It would also be most informative if you could provide me with a representative blot and details of the batch number. https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=7902&mode=questionaire I look forward to hearing from you.

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Question

Can we purchase RIPA buffer to lysate our cells from pig tissue. Thanks Dr. P. Sharma

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Answer

I'm sorry but we do not currently stock RIPA buffer.

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