Overview

  • Product name
    Mouse brain tissue lysate - total protein
    See all Brain lysates
  • Description
    Mouse brain total protein lysate
  • General notes


    Protein is isolated from whole tissue homogenates using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70ºC. Quality control: 1. The isolated protein pattern on SDS-PAGE gel is visualized by coomassie blue staining. The pattern is consistent with each lot. 2. The isolated protein is Western Blot analyzed by either a GAPDH or beta-actin antibody. The expression level is consistent with each lot.

  • Tested applications
    Suitable for: WBmore details

Properties

  • Form
    Liquid
  • Storage instructions
    Store at -80°C. Allow to warm to room temp and agitate gently before aliquotting.
  • Storage buffer
    Constituents: 1% Tergitol-NP40, Potassium chloride, 0.1% Bromophenol blue, Magnesium chloride, 1.2% Beta mercaptoethanol, 2% SDS, HEPES, Sucrose, 10% Glycerol

    With Protease inhibitor cocktail/EDTA
  • Concentration information loading...
  • Lysate notes
    Protein is isolated from whole tissue homogenates using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70ºC. Quality control: 1. The isolated protein pattern on SDS-PAGE gel is visualized by coomassie blue staining. The pattern is consistent with each lot. 2. The isolated protein is Western Blot analyzed by either a GAPDH or beta-actin antibody. The expression level is consistent with each lot.
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab30151 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. Samples need to boiled for 5 minutes and put on ice before use.

References

ab30151 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-5 of 5 Q&A

Answer

Thank you for contacting us. These two products are supplied as adult lysates, all older than 6 weeks , so they should be suitable for your studies.

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

Thank you for your helpful advice. I used Ponceau S on my most
recent blot and was able to visualize transfer of both my MW marker
and proteins in my cell lysate. It's certainly possible, however,
I could be getting over-transfer to some extent. I recently contacted
the lab that wrote the review for ab65166 on your website, and they
said that they performed a 16 h transfer at 20 V. With the new lysate,
my hope is to try both your one hour transfer conditions and the 16 h
transfer conditions and see if I see better transfer with either.
I'd also be open to trying higher MeOH and lower SDS content;
although, the aforementioned group apparently used a basic transfer
buffer with 20% MeOH and no SDS.
I'm not currently blotting for a loading control, but that is
a good suggestion. The best evidence I have for the success of my
WB protocol is that I could visualize Invitrogen's Precision
Plus MW marker after incubation with my secondary antibody.
In terms of lysing my own sample, I used the following reference to
prepare my lysis buffer (http://www.springerprotocols.com/Full/doi/
10.1007/978-1-61779-328-8_23?encCode=U0VOOjMyXzgtODIzLTk3NzE2LTEtODc5
&tokenString=k/NnQ1wdXIf1ihPjyV6QSQ==).
I certainly am open to try other lysis buffers but felt it might
be best to use a formulation that had been used successfully with
these channels before.
I very much appreciate the offer to send me another vial of ab30151.
From some initial reading, it looks like Nav1.6 expression increases
during maturation, so it's perhaps best to stick with the lysate
I'm already using. The order number for the original order is
xxx.
I can't thank you all enough for your help so far. Your team
has been incredibly responsive, and I very much appreciate your
assistance.

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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 xxx. The estimated delivery date is xxx as the item is currently backordered. I hope this is not causing a big problem. I apologize.

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

Thank you for your suggestions. In reviewing my previous blots, I
realized that all the ones I had run with the commercial cell lysate
had been stained with Amido Black, which made them unsuitable for
stripping and re-probing. I consequently ran another gel, loading
15 ug of ab30151 as well as the lysate for the system I'm trying
to study. I ran my transfer similar to before (75 V for 4 hours),
using 5% MeOH and 0.5% SDS in my transfer buffer, similar to your
suggestion. After transfer, I can see my MW marker (other company) fully transferred. I then pre-blocked with 3% BSA before
incubating with my primary antibody (ab65166) in 3% BSA overnight
at 4 oC. Finally, I incubated with the secondary antibody in 3%
BSA for 1 hour at room temperature and then did 3 10 minutes washes
with TBS-Tween before a final wash with TBS.
Upon visualization with my ECL+ kit, I only saw very high background
at moderate exposure times (1 minute) and couldn't see any signal
above background at very short exposure times (5-10 seconds). In
hindsight, I believe I should have incubated with the secondary
antibody in 10% milk.
I tried stripping the blot using the mild stripping protocol on
your website but still saw an almost equally strong background
upon visualization. At this point, I assume I must still have a
lot of secondary antibody non-specifically bound to the membrane,
so my next step is to try the harsh conditions.
Even though I see my full MW ladder transferred, I could definitely
believe that my transfer is still sub-optimal. I would like to try
alternative transfer conditions, but I have very little of my
remaining lot of ab30151 left. Would it be possible for me to obtain
another lot of this lysate at a discounted price? Alternatively, is
there another item in your catalog that you would recommend as a
positive control for the primary antibody that I could pay full price
for?

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Answer

Thank you for your reply.


I have a few additional suggestions. I am not sure if my colleague had mentioned those to you:

1) You can check the transfer with Ponceau S (which is a reversible membrane stain) to be sure that you transfered the proteins. This will give you also an additonal quality control for the transfer. My feeling is that transferingat 75V for 4 h could be too long. if the gel can withstand a higher voltage, I'd suggest to try 100V for an 1 h at 4 degree with stirring.

2) Further, I'd suggest to use 10% MeOH and reduce the amount of SDS to 0.1% in the transfer buffer. You want to have some SDS to prevent hydrophobic proteins (like Nav1.6) from precipitating in the gel during the transfer, but 0.5% seems a bit high.

3) Are you blotting for any other proteins, such as actin or tubulin as loading controls? This would be another way to check if the WB protocol in general works and if the mouse brain lysate you have is OK.

4) How are your samples lysed? I would recommend using RIPA buffer since the Nav1.6 protein is very hydrophobic.

I also checked online and found the following link for WB with membrane proteins:
http://www.sciencedirect.com/science/article/pii/S0003269708006829

Our WB protocol guide can be found here: https://www.abcam.com/index.html?pageconfig=resource&rid=11375and I have it also attached to this email.

To answer your question about the lysate:

I'd be happy to send you another vial of ab30151 free of charge. Alternatively, we also have ab7188 (Brain (Mouse) Tissue Lysate - normal tissue, 0 days old) [https://www.abcam.com/Brain-Mouse-Tissue-Lysate-normal-tissue-0-days-old-ab7188.html] in the catalog, but I am not certain if newborn mice express this protein strongly.
I can send you 1 vial of either one or the other free of charge. Please let me know which one you would prefer. Please let me know your order or PO number for the lysate.

I look forward to hear back from you and assist you further.

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Answer

Thank you for contacting us.
I am sorry to hear that the antibody might not be working as expected.

As we discussed over the phone, I would recommend the following positive control:

ab30151 (https://www.abcam.com/brain-mouse-whole-cell-lysate-normal-tissue-ab30151.html)

I hope this information is helpful to you. Please let me know what results you are getting with the positive control, and if the antibody can detect a band in this sample.

I look forward to hear back from you and wish you good luck.

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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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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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