Overview

  • Product name

    Proteasome Activity Assay Kit
  • Detection method

    Fluorescent
  • Sample type

    Cell Lysate
  • Assay type

    Enzyme activity
  • Assay time

    1h 00m
  • Species reactivity

    Reacts with: Other species, Mammals
  • Product overview

    Proteasome Activity Assay Kit (ab107921) takes advantage of the chymotrypsin-like activity, using an AMC-tagged peptide substrate (Proteasome Substrate (Succ-LLVY-AMC in DMSO), which releases free, highly fluorescent AMC (Ex/Em 350/440 nm) in the presence of proteolytic activity.


    The kit also includes a positive control (Jurkat Cell lysate with significant proteasome activity) and a specific proteasome inhibitor MG-132 which suppresses all proteolytic activity due to proteasomes. This permits differentiation of proteasome activity from other protease activity which may be present in samples.


    Proteasome activity assay protocol summary:
    - add samples, standards and positive control to wells
    - add proteasome inhibitor to half of sample and positive control wells
    - add proteasome substrate to sample and control wells
    - incubate for 20 min
    - analyze with microplate reader, incubate for further 30 min, analyze again

  • Notes

    The 20S proteasome assembly is the functional protease structure with chymotrypsin-like, trypsin-like and caspase-like protease activities

  • Platform

    Microplate reader

Properties

Images

  • Standard curve: mean of duplicates (+/- SD) with background reads subtracted

  • Proteasome Activity measured in Jurkat lysate

  • Proteasome Activity measured in HeLa lysate

  • Proteasome Activity measured in cell lysates showing activity (mU) per 1 mln cells.

    Samples with the concentration of 3.3e6 cells/mL were used. 50 μl of each sample was used.

Protocols

References

This product has been referenced in:

  • Merlos Rodrigo MA  et al. Proteomic Signature of Neuroblastoma Cells UKF-NB-4 Reveals Key Role of Lysosomal Sequestration and the Proteasome Complex in Acquiring Chemoresistance to Cisplatin. J Proteome Res 18:1255-1263 (2019). Read more (PubMed: 30592607) »
  • Hoffmann AC  et al. Extracellular aggregated alpha synuclein primarily triggers lysosomal dysfunction in neural cells prevented by trehalose. Sci Rep 9:544 (2019). Read more (PubMed: 30679445) »
See all 24 Publications for this product

Customer reviews and Q&As

1-10 of 23 Abreviews or Q&A

Proteasome activity in rat muscle

Good Good 4/5 (Ease of Use)
Abreviews
Soleus muscle was used.10mg of muscle was homogenized in 100µl of 0.5% NP-40, and 20µl of sample was used in duplicate in the plate.

Mr. Baptiste Jude

Verified customer

Submitted Oct 07 2016

Question
Answer

Start with 1 - 2 x 1e6 cells. Suspend the cell pellet in 500 μl (˜4 volumes) of the 0.5% NP40 solution on ice. Homogenize with a Dounce homogenizer (10 - 50 strokes) or equivalent on ice until efficient lysis is confirmed by viewing the cells under the microscope. Gentle sonication can be substituted for the Dounce homogenization, limited to several pulses while keeping the sample on ice. Spin down the sample at 13,000 rpm at 4C for 10-15 minutes. Use this supernatant for your subsequent assays.

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Answer

Thank you for your inquiry.

We do not recommend using samples prepared with RIPA buffer because the SDS tends to interfere with the enzyme.

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Answer

Addition of protease inhibitors for this assay (ab107921: Proteasome Activity Assay Kit) actually works well; since they will inhibit any proteases from degrading the proteins in the proteasome complex.

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Answer


Please use a white opaque plate. The troubleshooting guide (which recommends using a black plate for fluorescence) is just a general one.

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Answer

Thank you for contacting us.


I am pleased to answer your questions regarding the Proteasome Activity Assay Kit (ab107921).

1. I can confirm that it is necessary to create a new standard curve for every performed experiment. This will assure, that the preparation of samples and standards was done under the same conditions and makes it possible to compare samples with the standard results.

2. I can confirm, that it is possible to use cell pellets that were stored at -80 degrees as samples in this kit.

3. I can confirm, that 0.5% of NP-40 needs to be diluted in dH2O or PBS to homogenise the cells.

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 very much for your email.

As my colleague is not in the office today, I have looked into your inquiry.

I am happy to confirm that you can use any two time points as long as they are in the linear range. The difference between these points per minute will remain the same, as it is in the linear range. You can try the to calculate with two different time sets to confirm. The importance of choosing them, is that they are in the linear range - otherwise there are no rules. I would chose two time points which lay relativelyapart, just to reduce noise. This would be the first time point near the end of the linear range (however for sure still in the linear range) and the second time point in the beginning of the linear range (however again, for sure in the linear part of the graph).

Feel free to test the calculation with different time pairs in the linear range as well to become confident of the reading.

I hope this information is helpful. Please do not hesitate to contact us again, should you have any further question or concern.

I wish youa good week.

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Answer

The standard graph looks good. Based on that,you are right in saying that the samples can be diluted further maybe 1:5 this time for better time points.
From this run, assuming that the X axis is time, I think maybe 8 and 10 (units unknown) would fit most samples. But again those times are very close apart and don’t fit all samples.

Yes, proteasome substrate must be added to all wells (page 8 of the https://www.abcam.com/ps/products/107/ab107921/documents/ab107921%20Proteasome%20Activity%20Assay%20Kit%20(Website).pdf).

I hope this is helpful.

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Answer


Could you please send the standard curve for the attached plot?
My colleague would be able to help with the T1 and T2 looking at the curve.

Alternatively, this is the way you could decide the time points:
It is essential to read RFU1, iRFU1, RFU2 and iRFU2 in the linear reaction range.
It will be more accurate if you monitor the reaction kinetics as shown in Fig. B, and then choose T1 and T2 in the appropriate linear range. From our experience, initial readings RFU1 and iRFU1 should be measured after ˜ 20 - 25 min.

Please don’t add the inhibitor to any standard wells. Use only the assay buffer as your blank.

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Answer

I just wanted to let you know that I am still working on your inquiry. I have contacted the originator of this product for the information you requested, and I am awaiting a reply.

I am very sorry for the delay. I will forward to you the information as soon as I receive it. Thank you for your patience.

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1-10 of 23 Abreviews or Q&A

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