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Mammalian Cell Extraction Kit (ab65399)

Reviews (1)Q&A (3)References (1)

Key features and details

  • Assay type: Direct
  • Assay time: 20 min
  • Sample type: Adherent cells, Suspension cells, Tissue

Overview

  • Product name

    Mammalian Cell Extraction Kit

  • Sample type

    Tissue, Adherent cells, Suspension cells

  • Assay type

    Direct

  • Assay time

    0h 20m

  • Product overview

    Abcam's Mammalian Cell Extraction Kit provides optimized cell extraction buffer, protease inhibitor cocktail, and DTT for convenient extraction of mammalian proteins from cultured cells and tissue samples, under nondenaturing conditions. Cell lysate prepared using the kit can be used in a variety of applications, such as enzyme activity assays (e.g. caspase activity assays), Western blot analysis, and others. The entire procedure takes less than 20 minutes.
    Visit our FAQs page for tips and troubleshooting.

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K269 Mammalian Cell Extraction Kit. K269-500 is the same size as the 500 test size of ab65399.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

Properties

References (1)

Publishing research using ab65399? Please let us know so that we can cite the reference in this datasheet.

ab65399 has been referenced in 1 publication.

  • Caglayan C  et al. Rutin ameliorates mercuric chloride-induced hepatotoxicity in rats via interfering with oxidative stress, inflammation and apoptosis. J Trace Elem Med Biol 56:60-68 (2019). PubMed: 31442956

Customer reviews and Q&As

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1-3 of 3 Q&A

Question

A customer asks an information about ab65399 Mammalian Cell Extraction Kit. He will perform extraction from colon tissues and want to know a exact protocol for this. How much (gram) tissue must be used with how much lysis solution in the kit? (On the protocol booklet, It doesnt write about tissue weight)

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Answer

For tissue samples: Start with 10-100 mg of the tissue, add 500-1000 μl (or ~4-6 volumes) of the assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Use the eluate for your subsequent assays. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.

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Question

Dear Ms or Mrs,

Thank you very much for your patient explanation !

May I ask some new questions about this product:

1.We want to extract the proteins include membrane proteins and nuclear proteinsfrom rat testes, Can we use this product to extract both the membrane and nuclear proteins at the same time ?

2. In order to smash the tissues,the tissuesare alwayssonicated by the homogenate machine, so can we use your product to smash the tissues by this way ?

Thank you again for your help ! My reference number is CCE3575230

best regards !

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Answer

I double checked with the lab to answer this question.

1. I am happy to confirm that this kit can be used to isolate both membrane and cytoplasmatic proteins. The membrane proteins are in the pellet from step 5. If you would like to also isolate the membrane proteins: DO NOT discard the pellet from step 5.

But resuspend the pellet in PBS containing NP-40 or Triton 20 (0.1%).

2. I can confirm that sonication is possible method to generate the cell homogenate for the kit. This has not be experimentally tested by us, but shouldbe suitable for this kit.

I hope this information is helpful and wish you good luck with your experiments.

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Question

To whom may concerns,

Thank you for your attention.

I am seeing the product named 'mammalian cell extraction kit',its item is ab65399fromabcam.There has no protocal about this product , I am just wondering toask some questions :

1. I saw that the product can be used for tissue samples, we have rat tissues saved at -80 degrees ,can we use this product to extract proteins for western blot?

2. We always use the homogenate machine to smash the tissues before the extraction of the proteins , so can we use your product to smash the tissues ? do you think it is Ok or not for us to smash the tissuesin this way ?

3. Do you have some recommend protocals ?

Looking forward to your reply ! Thank you for your help !

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Answer

Thank you for your inquiry.

Please find the protocol for this kit below:

Mammalian Protein Extraction Protocol

A. General Consideration and Reagent Preparation:

- After opening the kit, store Cell Extraction Buffer at +4oC. Store Protease Inhibitor

Cocktail and DTT at –20oC.

- The Protease Inhibitor Cocktail is provided as lyophilized form. To reconstitute,

add 100 μl DMSO to the vial, pipet several times to dissolve all powder (This

makes 500X concentrated Protease Inhibitor Cocktail).

- Before use, add 2 μl of DTT and 2 μl of Protease Inhibitor Cocktail to 1 ml of Cell

Extraction Buffer (The mixture is referred as Extraction Buffer Mix).

- Be sure to keep the Extraction Buffer Mix on ice at all times during the experiment.

- The following protocol is described for extraction of ˜2 x 106 cells and should

generate ˜100-300 μg of cell lysate. If large amount of cell lysate are desired,

scale up the volumes proportionally.

B. Mammalian Protein Extraction Protocol:

1. Collect cells by centrifugation at 600 x g for 5 minutes at 4oC.

Note: For adherent cells, scrape cells into PBS and spin down to pellet cells.

2. Resuspend cells in 100 μl of the Extraction Buffer Mix. Pipet up and down

several times.

Note: For tissue samples, homogenize tissues in 2-3 volume of the

Extraction Buffer Mix, until it is completed lysed.

3. Incubate on ice for 10 minutes, then vertex for 5 seconds.

4. Centrifuge in a microcentrifuge at top speed for 3 minute.

5. Collect the supernatant (Cell Lysate) and discard the pellet.

6. Store cell lysate at –70oC for further studies.

Please follow this protocol for tissue samples. I can confirm that your homogenate machine will be suitable for the purpose of generating WB lysates.

I hope this information is helpful and wish you good luck with your experiments.

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