Overview

  • Product name
    Jurkat whole cell lysate
    See all Jurkat lysates
  • General notes
    Cell line: Jurkat, clone E6-1 (Human acute T cell leukemia).
    Growth media: RPMI and 10% NCS (Newborn calf serum).


    Jurkat cell lysate was prepared by homogenization in modified RIPA buffer (150 mM sodiumchloride, 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
    Jurkat cell lysate was prepared by homogenization in modified RIPA buffer (150 mM sodiumchloride, 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
    Often this cell line is called 'JM' (Jurkat and JM are derived from the same patient and are sister clones), occasionally JM may be a subclone with somewhat divergent features. Jurkat cells can be transfected and are therefore useful for studies of blood proto-oncogenes expression, apoptosis and cell survival (e.g HIV). They produce IL-2 and can also be used for studies of differentiation.

Applications

Our Abpromise guarantee covers the use of ab7899 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. Jurkat cell lysate is ready to load on SDS-PAGE for Western blotting, 20 µg per lane is recommended for mini gel.

References

This product has been referenced in:
  • Berra-Romani R  et al. Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture. Am J Physiol Cell Physiol 295:C779-90 (2008). Read more (PubMed: 18596214) »
See 1 Publication for this product

Customer reviews and Q&As

1-8 of 8 Q&A

Question
Answer

Thank you for sending this data to me.

While reviewing this slide, I noted that your bands are white. Oversaturation of secondary antibodies can lead to masked signal or false positive. In this case, the secondary antibody which you are using at 1:2500, ab6721 has been used successful at dilutions at almost 100 times what you have used. This antibody can be diluted to 1:48,000 to 1:207,000 in many instances. I would recommend to titer this secondary antibody to find the optimal dilution for this product. As your bands are super saturated at 1:2500 I would recommend a titer range of ab6721 beginning at 1:10,000.

Hopefully by controlling the secondary antibody saturation, the true signal from these anti-PDGF antibodies can emerge.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

Thank you for contacting us.

I was not able to view the attachment included. Would you be able to re-send this to me as a jpeg, ppt or png file? Also could you let me know some of the details of the experiement (amount of positive control used, ab concentration,etc)?

I look forward to your reply. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question
Answer

Thank you for confirming these details and for your cooperation. I am sorry that you are continuing to have difficulties when using these products. As requested, I have issued a free of charge replacement of ab73229 PDGF BB Protein and have arrange for our Jurkat Cell lysate (ab7899) to be sent to you free of charge as well. The order number for these products is xxxx. To check the status of the order please contact our Customer Service team and reference this number. I do hope that by increasing the amount of this replacement protein to 100ng/well, by using the Jurkat lysate at 30ug/well a second control and by using an increased concentration of ab135881 to 1:500 as seen on the datasheet, you will be able to see good results. 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.
According to the datasheet, the this protein is not expressed in most lymphoid cell lines: B-cells, T-cells and NK cells. Therefore, I would suggest to use a T-cell line like Jurkat lysate as a negative control.

https://www.abcam.com/index.html?datasheet=7899 https://www.abcam.com/index.html?datasheet=7899.

As a "negative control" for WB I woul suggest to perform a non-primary control, which simply means skipping the promary antibody incubation.

I hope this information is helpful to you. 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

BATCH NUMBER 144208 ORDER NUMBER PO#57883 DESCRIPTION OF THE PROBLEM Using anti-Lef-1 antibodies purchased from [ a competitor] (same as Ab12035 and Ab12034) and the Using Jurkat whole cell lysate as a control, I was assuming I would see a 55-57kD band on a western blot. On a 4-20% gradient gel, I see a predominant band at 125kD. There are also minor bands all of equal intensity ranging from125kD down to 15kD. None of these bands stand out. The western itself looks quite good, the gel ran well, good separation, etc... I don't trust the Jurkat positive control if nothing is standing out at the predicted molecular weight of 55-57 kD. What is the major band I am seeing in the Jurkat whole cell lysate at 125 kD? PRIMARY ANTIBODY [ a competitor] (Mab 3752) and [ a competitor](Mab3751) in Blocking buffer 1 hour RT (use at 5ug/ml) Wash with blocking buffer. DETECTION METHOD SuperSignal West Pico Chemiluminescent Substrate kit from Pierce. POSITIVE AND NEGATIVE CONTROLS USED Jurkat whole cell lysate (abcam) RIPA e 9.5 day embryos lysate -- SAMPLE M-PER Mammalian Protein Extraction Reagent- e 9.5 day embryo lysate- SAMPLE ANTIBODY STORAGE CONDITIONS This is not an antibody. It is the positive control for an anti-Lef-1 antibody. I have now purchased 3 tubes of Jurkat whole cell lysate from Abcam. I have tried to get the anti-lef-1 antibodies to work in a western using the Jurkat whole cell lysate as a positive control. I am supposed to see at 55-57 kD band. SAMPLE PREPARATION The Jurkat whole cell lysate ( (ab7899) comes ready to load. AMOUNT OF PROTEIN LOADED 10 ul of a 2 mg/ml sample...20 ug of whole cell lysate...as recommended on the product sheet ELECTROPHORESIS/GEL CONDITIONS Reducing conditions 4-20% gradient gel from Bio-Rad TRANSFER AND BLOCKING CONDITIONS transfer 2 hours at 70 volts Block with TBS/0.05% Tween 20/3% non-fat dry milk (Bio-Rad) 1 hour RT SECONDARY ANTIBODY Abcam (Ab6728) Rabbit anti-mouse IgG H&L (HRP) Tried orinally at 1:10,000. Very minimal signal. Used last time at 1:4000. Good signal minimal background. Diluted in Blocking buffer 1:4000 RT 1 hour Wash in blocking buffer. Last few washes in TBS before detection. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Washes -first time concentration of Tween probably too high

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Answer

I'm sorry to hear you are experiencing problems with our Jurkat lysate ab7899 and have tried to look at the [ a competitor] antibodies to see if they were the same as ab12034 and ab12035. In fact they are not the same antibodies but the same clones, and the [ a competitor] antibodies come as "purified ascites" while the Abcam antibodies have been Protein G purified. It is likely that the purification process is therefore different and ascites will give more bands as it is not well purified. I would like to recommend asking [ a competitor] regarding that reported 125kda band with the antibodies, as this could be due to the purity of their products. I suspect the problem could also be due to the blocking agent in the experiment, especially if you see lots of non specific bands with your other lysates. We find that a number of antibodies bind non specifically if too much blocking agent is present, I would therefore recommend trying a 1hr incubation in milk 5% in TBST on a membrane and a one hour incubation in BSA 5% in TBST on another membrane. Then I recommend to wash lightly the membranes in TBST (10seconds) and incubate the primary antibody (and secondary) in TBST only. I hope these suggestions will help you, please do not hesitate to contact me if I can be of further help,

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Question
Answer

Please let me know what extra information you require. This product is a Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate which we sell in units of 100µg in 1 x SDS sample buffer containing 5% b-mercaptoethanol. There is further information about the cell line in the relevance section of the datasheet. I'm not sure what extra information you require.

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Answer

Thank you for your email and I'm sorry to hear about the difficulty you are experiencing with ab4788. I apologize for the shortage that you received and can certainly send you a replacement vial free of charge. Can you please tell me the batch number that you received (it is located on the vial)? Also, what was the Abcam order number or purchase number that was used? Thank you, and I look forward to hearing from you.

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Answer

Yes, Jurkat cell lysate would be a good positive control for active caspase 9. Jurkat 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.

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