Proteasome Activity Assay Kit (ab107921)

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.

Thank you for your inquiry.

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

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.

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

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.

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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.

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.

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.