Overview

  • Product name
    HeLa nuclear extract lysate
    See all HeLa lysates
  • General notes
    We recommend aliquoting the extracts into single-use fractions and then storing them.
  • Tested applications
    Suitable for: WBmore details

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped on dry ice. Upon delivery aliquot and store at -80ºC. Avoid freeze / thaw cycles.
  • Storage buffer
    pH: 7.90
    Constituents: 0.48% HEPES, 20% Glycerol, 0.01% Magnesium chloride, 0.077% DTT, 0.004% EGTA, 2.05% Sodium chloride, 1% NP-40
    Note: With Protease inhibitors
  • Concentration information loading...
  • Research areas
  • Background
    HeLa cells are human epithelial cells from a fatal cervical carcinoma. The cell line was derived from cervical cancer cells taken from Henrietta Lacks, in 1951. Horizontal gene transfer from human papillomavirus 18 (HPV18) to human cervical cells created the HeLa genome which is different from either parent genome in various ways including its number of chromosomes. HeLa cells have a modal chromosome number of 82, with 4 copies of chromosome 12 and 3 copies of chromosomes 6, 8, and 17. HeLa cells are adherent cells (they stick to surfaces) and maintain contact inhibition in vitro.

Applications

Our Abpromise guarantee covers the use of ab14655 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.

References

This product has been referenced in:
  • Weitzel LR  et al. Discovery and verification of protein differences between Er positive/Her2/neu negative breast tumor tissue and matched adjacent normal breast tissue. Breast Cancer Res Treat 124:297-305 (2010). WB . Read more (PubMed: 20087651) »
See 1 Publication for this product

Customer reviews and Q&As

1-10 of 10 Q&A

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 xxxxx. Included in this order are positive control lysates for these products. 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

Hello, I’m sending pictures of my blots because I’m unsure of what band is my protein of interest. Can you point out the correct band in my blots? The following is a description of what I did: Samples The samples are lysate fractions from A375 melanoma cells that have been exposed to different doses of UVB light. Zero is the untreated control. Using the “Nuclear Fractionation Protocol” from the Abcam website (using buffers consisting of HEPES, salt, +/- 26% glycerol) I made cytosolic and nuclear fractions. For the protocol, I used a 25 gauge needle instead of a Dounce homogenizer. I measured the concentration using a standard BSA assay and loaded 30ug per lane. Positive Control 20ug of HeLa nuclear lysate also purchased from Abcam (ab14655) Western Blot I ran the samples in the following order for each antibody: 1) HeLa nuclear lysate, 20ug 2) Untreated cells, cytosol fraction, 30ug 3) Untreated cells, nuclear fraction, 30ug 4) Treated cells, 30mJ/cm2, nuclear fraction, 30ug 5) Treated cells, 50mJ/cm2, nuclear fraction, 30ug 6) Treated cells, 80mJ/cm2, nuclear fraction, 30ug These samples were diluted in standard sample buffer and ran on a 10% SDS gel. Transfer to nitrocellulose membrane was done using a semi-dry transfer apparatus. The membrane was rinsed in 0.5% Tween-PBS (PBST) briefly before incubating in a 5% BSA/PBST blocking solution for at least 1 hr at room temp on a rocker. The blocking solution was made using dry BSA. The membrane was briefly rinsed 3x in PBST before incubation with the primary antibody diluted as follows in 0.25% BSA/PBST. ab13537 = DNMT1. 1:1,000 ab13888 = DNMT3A. 1:750 ab13604 = DNMT3B. 1:750 The incubation was overnight (at least 14 hr) at 4 degrees on a rotator. The membranes were briefly rinsed 3x and then washed 3x in PBST. The secondary antibody is rabbit anti-mouse IgG+A+M(H+L) conjugated with HRP diluted to 1:10,000 in 0.25% BSA/PBST. The membranes were briefly rinsed 3x, washed 2x in PBST, washed once in PBS, and incubated with standard ECL components before exposure to blue x-ray film. Exposure times ranged from approx. 30 sec to 1 min. Note: much the molecular weight marker faded away after the washes in DMNT-3A,-3B which were done simultaneously.

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Answer

Thank you for contacting us.

I would like to thank you for sending me your protocol, this information is a great help.

I did not recieve any images with you email however. Could you please resend those to me?

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

Hello,



I’m sending pictures of my blots because I’m unsure of what band is my protein of interest. Can you point out the correct band in my blots?



The following is a description of what I did:



Samples

The samples are lysate fractions from A375 melanoma cells that have been exposed to different doses of UVB light. Zero is the untreated control. Using the “Nuclear Fractionation Protocol” from the Abcam website (using buffers consisting of HEPES, salt, +/- 26% glycerol) I made cytosolic and nuclear fractions. For the protocol, I used a 25 gauge needle instead of a Dounce homogenizer. I measured the concentration using a standard BSA assay and loaded 30ug per lane.



Positive Control

20ug of HeLa nuclear lysate also purchased from Abcam (ab14655)



Western Blot

I ran the samples in the following order for each antibody:

1) HeLa nuclear lysate, 20ug

2) Untreated cells, cytosol fraction, 30ug

3) Untreated cells, nuclear fraction, 30ug

4) Treated cells, 30mJ/cm2, nuclear fraction, 30ug

5) Treated cells, 50mJ/cm2, nuclear fraction, 30ug

6) Treated cells, 80mJ/cm2, nuclear fraction, 30ug



These samples were diluted in standard sample buffer and ran on a 10% SDS gel. Transfer to nitrocellulose membrane was done using a semi-dry transfer apparatus. The membrane was rinsed in 0.5% Tween-PBS (PBST) briefly before incubating in a 5% BSA/PBST blocking solution for at least 1 hr at room temp on a rocker. The blocking solution was made using dry BSA.



The membrane was briefly rinsed 3x in PBST before incubation with the primary antibody diluted as follows in 0.25% BSA/PBST.

ab13537 = DNMT1. 1:1,000

ab13888 = DNMT3A. 1:750

ab13604 = DNMT3B. 1:750

The incubation was overnight (at least 14 hr) at 4 degrees on a rotator.



The membranes were briefly rinsed 3x and then washed 3x in PBST. The secondary antibody is rabbit anti-mouse IgG+A+M(H+L) conjugated with HRP diluted to 1:10,000 in 0.25% BSA/PBST.

The membranes were briefly rinsed 3x, washed 2x in PBST, washed once in PBS, and incubated with standard ECL components before exposure to blue x-ray film. Exposure times ranged from approx. 30 sec to 1 min.



Note: much the molecular weight marker faded away after the washes in DMNT-3A,-3B which were done simultaneously.

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Answer

Thank you for contacting Abcam.

I have attached the ppt slide with a red arrow next to the bands that I think are the correct bands(they are the same that you have marked).

There are a few protocol changes that I would recommend to reduce the background that you are seeing:

1 - Use an 8.5% gel instead of 10% and run the gel out further so that you loose the lower portion of the gel, as this will allow you to get greater separation and clearly see your band of interest.

2 - Reduce the total amount of protein that you are loading to 25ug.

3 - Increase the duration and number of washes, to at least 4 x 5 minutes and also use up to 0.2%Tx-100.

4 - Use 1% BSA instead of0.25% BSA in your primary antibody incubation buffer.

5 - Reduce the primary antibody concentration to 1/1250 or 1/1500

Hopefully the above changes will allow you to get a cleaner signal, but I am confident that the antibody is recognizing the correct protein.

If there is anything else I can help you with, please let me know.

.

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Answer

Thank you for your reply.

I am sorry that we were not able to resolve the issues you were having with ab14655 and I have processed your request for a refund. Your credit note number is ******.

Please let me know if there is anything else I can help you with.

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Answer

Thank you for your reply.

The product ab29545 is a whole cell lysate not a nuclear lysate. The only nuclear lysate we have in stock is the ab14655that you currently have.

If you still wish for me to send you ab29545, the whole cell lysate, please let me know and I will have it sent today. Otherwise, I can refund the cost of ab14655, as it did not produce a signal for you.

I look forward to your reply and resolving this issue for you.

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Answer

Thank you for that information.

I do not think adding 4ul more will increase your signal and since you can only load a maximum of 22ul/well, I would like to send you ab29545 as a free of charge replacement. This HeLa cell lysate is already in sample buffer and is at a higher concentration that the original one sent to you, so you can load more total protein and hopefully see a signal with your 3 antibodies. If this is ok with you, please let me know and I will process the replacement for you.

I look forward to you reply.

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Answer

Thank you for your reply.

I appreciate your patience in this matter, but when you say you loaded 2.5mg/ml of total protein, how many microlitres of lysate is that? I ask, as we send 100ug of total protein at at concentration of 2.5mg/ml (therefore we you should have received 40ul total volume). I just want to make sure that the problem is with the lysate and not that more needs to be used. Although you used both the HeLa and PC12 lysates at the same concentrations, it may be that you need to use more of the HeLa lysate, as you were seeing a signal for Tubulin (the signal was weak, but it was present).

Once again thank you for your patience in this matter, if it does turn out that there is a fault with the lysate, then I would be more than happy to send a new vial.

I look forward to your response.

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Answer

Thank you for contacting Abcam.

I am sorry about the problems you have been having with ab14655, HeLa cell lysate. I was wondering if you could tell me how much total protein you loaded of the cell lysate. I ask as there does seem to be a signal (a weak signal) for Tubulin and I am wondering if you load more of the lysate onto the gel, you would increase the signal for Tubulin and also begin to see a signal for the other antibodies.

If increasing the total amount of protein in the well does not improve the signal, then I would be happy to send you another aliquot of the HeLa lysate.

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

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Answer

Thank you for your enquiry. The nuclear extracts were prepared as detailed in: Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucl. Acids Res. 11, 1475–89. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. To prepare a nuclear lysate from HeLa cells I would recommend that you use RIPA buffer as follows. RIPA buffer (RadioImmunoPrecipitation Assay) buffer: RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent and is particularly used for nuclear membrane disruption for nuclear extracts. RIPA buffer gives low background but can denature kinases. It can also disrupt protein-protein interactions (and may therefore be problematic for immunoprecipitations/pull down assays). 50mM Tris HCl pH 8 150 mM NaCl 1 % NP-40 0.5 % sodium Deoxycholate 0.1 % SDS The 10 % sodium deoxycholate stock solution (5 g into 50 ml) must be protected from light. The 100 mM EDTA stock solution is made with 1.86 g into 40 ml H2O and then add NaOH to dissolve and adjust pH to 7.4. Finally, adjust the total volume to 50 ml). Store the buffer at 4°C. Please do not hesitate to contact me should you require further assistance.

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