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  1. Link

    products/tissue-lysates/nih3t3-nuclear-extract-lysate-ab14874.pdf

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NIH/3T3 nuclear extract lysate (ab14874)

  • Datasheet
  • SDS
Submit a review Q&A (6)

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Preparation
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Nuclear Extraction Kit (ab113474)
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Nuclear Extraction Kit - Soluble / Insoluble Fractions (ab219177)

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Overview

  • Product name

    NIH/3T3 nuclear extract lysate
    See all NIH 3T3 lysates
  • General notes

    Cell line: NIH/3T3 (Mouse embryonic fibroblast).
    Extracts have been quality control tested by Western blot and the Electrophoretic Mobility Shift Assay (EMSA).

Properties

  • Mycoplasma free

    Yes
  • Form

    Liquid
  • Storage instructions

    Aliquot and store at -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer

    pH: 7.90
    Constituents: 0.75% Potassium chloride, 0.009% PMSF, 0.01% Magnesium chloride, 0.008% DTT, 0.48% HEPES, 20% Glycerol
  • Concentration information loading...
  • Research areas

    • Kits/ Lysates/ Other
    • Lysates
    • Nuclear Lysates
    • Mouse
    • Other
  • Background

    NIH 3T3 cells are established from a NIH Swiss mouse embryo. These cells are highly contact inhibited and are sensitive to sarcoma virus focus formation and leukaemia virus propagation. Cells have now lost their contact inhibition. This cell line was established from NIH Swiss mouse embryo cultures in the same manner as the original random bred 3T3 and the inbred BALB/c 3T3. The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays. It is therefore used for DNA transfection studies

Protocols

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

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab14874? Please let us know so that we can cite the reference in this datasheet.

ab14874 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

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1-6 of 6 Abreviews or Q&A

Question

Hello,

Yesterday, I used your p19ARF antibody(ab80) for my samples. Also I set NIH3T3 whole cell lysate as a positive control, but it didn’t work. (no band.)

I borrowed this cell line from other lab. After I cultured, I extracted lysate by myself.

I think there is some possibility that my NIH3T3 had any problems. So, I plan to order your cell lysate and check some mouse embryonic fibroblast cell line; ab7179,ab14874,ab7901,ab3874.

I’d like to ask you which is the best as positive control for p19ARF antibody? Or is there no difference among them?

Thank you,

Read More

Abcam community

Verified customer

Asked on Oct 11 2012

Answer

Thank you very much for contacting us with your enquiry.

I'm sorry to hear that there were no bands in Western blot using this antibody. I would expect any of the MEF cell lines to work as a positive control, but I suggest using a whole cell lysate (ab14874 is a nuclear fraction) since that is what we have tested with ab80. What kind of samples are you using other than the MEF lysate?

If you'd like to send some more details about your protocol ( how much protein was loaded, dilution of antibody, blocking solution, etc) I'd be happy to take a look and see if we have any suggestions to improve the results.

I look forward to hearing from you. If there's anything else that we can do for you, please let me know and I'll be happy to help.

Read More

Abcam Scientific Support

Answered on Oct 11 2012

Question

Thanks very much for your prompt reply and for checking for possible related proteins. For your questions:

How much lysate per lane? 30 ug in the first lane, 15 ug in the second (indicated at the top of the blot)

Blocking solution? Milk: GE, RPN 418V, 2% in TBS/0.1% Tween-20

Exposure time? One minute

Lysate storage/handling? Lysate was stored at -80 degrees, thawed, combined with equal volume of loading buffer (BioRad 161-0737 with 5% beta-mercaptoethanol), boiled 5 minutes, spun down briefly before loading.

Thanks very much.

Read More

Abcam community

Verified customer

Asked on Aug 31 2012

Answer

Thank you very much for your reply and for the additional information.

Have you tried any other exposure times? Since the specific band seems to be the strongest (with 1:100 of antibody and 15-30 ug of protein), you can probably reduce the exposure time, which will help reducebackground as well. I'd also recommend using a 5% milk block to further reduce non-specific binding. If these suggestions don't improve the results, I'd be happy to send a replacement antibody or issue a credit or refund if you'd like.

Please keep me updated about any new results, and if you have any questions or need anything else let me know and I'll be happy to help.

Have a nice day.

Read More

Abcam Scientific Support

Answered on Aug 31 2012

Question

Hi--I recently ran a Western with OGG1 antibody ab91421 and 3T3 nuclear lysate ab14874. I got the bands near 39 & 60kD as shown in

https://www.abcam.com/Ogg1-antibody-ab91421.html

but also many other bands (below). Do you have suggestions as to what these others are, besides your general notes in

https://www.abcam.com/index.html?pageconfig=resource&rid=11352

and

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

Thank you!

Read More

Abcam community

Verified customer

Asked on Aug 30 2012

Answer

Thank you very much for contacting us with your question and for sending the image of your results.

According to the UniProt page for this protein, there aren't any known post-translational modifications like cleavage or glycosylations that might result in multiple bands-

http://www.uniprot.org/uniprot/O08760

The human protein is known to have several isoforms, but the C-terminal regions are very different and probably wouldn't react with the antibody ab14874. So, these other bands are most likely non-specific. I also ran a BLAST of the immunogen region against the mouse proteome, and no proteins other than OGG1 are predicted to react with the antibody based on these results. Due to this information, we are not sure of the identities of these extra bands, but there may be ways to reduce them.

Would you mind telling me a bit more about the protocol that was used?

1) How much lysate was loaded per lane?
2) What kind of blocking solution was used?
3) How long was the exposure time?
4) How was the lysate stored before being loaded onto the gel?

I look forward to hearing from you. We do fully guarantee the results of this antibody, so I can send a replacement antibody or issue a credit or refund if the results are not satisfactory. Please let me know if you have any questions or if there is anything else that we can do for you.

Read More

Abcam Scientific Support

Answered on Aug 30 2012

Question

Do you have any Ezh1 antibody that works well on mouse tissue?

Read More

Abcam community

Verified customer

Asked on Mar 20 2012

Answer

Thank you for contacting us. We have three antibodies that may be suitable for your purposes:

ab13665: rabbit polyclonal to EZH1 validated in WB on mouse and human samples
ab86128: rabbit polyclonal to EZH1 validated in WB and ICC/IF on mouse and human samples
ab64850: rabbit polyclonal to EZH1 validated in WB, IHC-P, and ELISA on human and mouse samples

Please let me know if you need any additional information or assistance.

Read More

Abcam Scientific Support

Answered on Mar 20 2012

Question

I ran a western blot with your cell lysate and blotted with Ezh1 Ab at
1:5000 and then Donkey anti-RHRP 1:100,000 dilution. The whole blot turned
black after 1 s exposure time.
I tried to figure out which reagent causing the high background. I used 3
different blocking solutions (5% milk, BSA and Donkey serum) to block a
piece of PVDF (no protein on) respectively. I incubated each blot either
in secondary RHRP (1:10,000)or Ezh1 (1:5000). For the Ezh1 blots, I washed
it as you suggested and then blotted with RHRP . For the RHRP only blots,I
developed the film after washing steps.
The results showed that My secondary antibody does not interact with 5%
milk. However, there is something in your Ezh1 antibody interacting with
5% milk (WB is attached). Is there any other blocking solution I can use?
Or it is because I just got bad luck to get a bad batch. I know this Ezh1
should work based on literature. Let me know what you think.

Read More

Abcam community

Verified customer

Asked on Mar 09 2012

Answer

Thank you for sending the image of your blots.

When you use the PVDF membrane, are you pre-wetting it with methanol? Because PVDF is hydrophobic, it may not properly bind the blocking agent without the pre-wetting step. This could potentially cause the black blot you are seeing.

The speckled signal observed with the donkey anti-rabbit HRP secondary and 5% donkey serum blocking agent is typically a product of contamination in the blocking buffer or primary antibody diluent. This may be prevented by either filtering your reagents or using fresh buffers. This could also bea result of contamination on the sponge in your transfer apparatus. Thoroughly washing the transfer equipment should prevent this.

This EZH1 antibody is validated using 5% BSA in TBST as a blocking agent, but it should be suitable for use with milk and donkey serum as well. Because the signal observed is so strong, I would recommend using a much higher dilution of secondary antibody (at least 1:50k - 1:100k). Increasing the blocking time to overnight at 4C may also help. If you are looking to try an alternative blocking agent, gelatin is sometimes used. There are also several commercial blocking agents available (including ab126587 and ab64234), however these tend to be casein based. Because casein is the major protein in milk, they may or may not produce different results in your experiment.

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

Read More

Abcam Scientific Support

Answered on Mar 09 2012

Question

My WB membrane is completely black using ab64850.

Read More

Abcam community

Verified customer

Asked on Mar 05 2012

Answer

Thank you for your phone call. As we discussed, it is possible that your 6 x 15 minute washes are washing the blocking agent off your membrane. It may help to reduce the wash time to 3 x 5 min, shorten the primary antibody incubation to 1 hour at RT, and use less primary antibody. I hope this will help to improve your results, if not, please let me know and I will be happy to help you further.

Read More

Abcam Scientific Support

Answered on Mar 05 2012

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