Product Review: UCHL1 antibody - C-terminal region (ARP59285_P050) with cytosolic protein extracts from rat brain for Western Blot

UCHL1 (ARP59285_P050) Western Blot
UCHL1 (ARP59285_P050) cytosolic protein extracts from rat brain

"This antibody provides a specific and very robust signal when using brain material."

Data submitted by: Andreia Carvalho, Instituto de Biologia Molecular e Celular (IBMC), Portugal


1. What applications did you test Aviva’s reagents in?

Western blot analysis

2. How many different experimental trials were conducted?

3 trials with cytosol prepared from male rat brain and rat liver and HeLa cells.

3. Were controls were used in your experiment? If so, what were they?

Yes. We have included in our analysis protein extracts incubated with HA‐UbVME. HA‐ UbVME  is  an  ubiquitin‐based  electrophilic  probe  that  modifies  covalently  DUBs  that belong to the cysteine protease family (Borodovsky A Chem Biol 2002). When modified with HA‐UbVME, DUBs suffer a 10‐kDa increase in their molecular weight. We have also included in our analysis brain material from rat. UCHL1 is 1% of total protein content.

4. Can you please provide the protocol for your application procedure?  Please include all relevant info (dilutions, fixatives, etc.).

HeLa cell total extracts were prepared by sonication in SEM buffer (0.25 M sucrose, 20 mM MOPS‐KOH, pH 7.2, 1 mM EDTA‐NaOH, pH 8.0). Male rat liver/brain cytosol was prepared using the protocol described recently (Grou CP JBC 2012).  Each protein extract (5 μg for rat brain cytosol, 100 μg for rat liver cytosol and HeLa cells) was incubated in the   absence  or  in  presence  of  1  μM  HA‐UbVME  for  15  min  at  25  ºC.  Reactions  were terminated by the addition of Laemmli sample buffer. Samples were analyzed on a 12% SDS‐PAGE and blotted onto a nitrocellulose membrane.

The membrane was stained with Ponceau S to assess protein loadings. After destaining with TBS1x, the membrane was blocked with 5% skimmed (SM)/ TBS1x for 1h at room temperature (RT), and subsequently incubated overnight at 4 ºC with a 1:1000 dilution (1 μg/ml) of the primary antibody in 5% SM/TBS1x . The membrane was then washed with TBS1x/Tween 20 0.1% (2x 5 min, RT), and incubated with an alkaline phosphatase‐ conjugated secondary antibody (anti‐rabbit, Sigma) for 1h at RT in 5%/TBS1x. The washing procedure was repeated, followed by incubation with developing agents NBT/BCIP (BioRad).

5. How would you rate this antibody on a scale from 1‐5? Why?

Classification: 4. This antibody provides a specific and very robust signal when using brain material.

6. Would you use this antibody in future experiments or to publish your data? Why or why not?

Yes. This antibody provides a specific and very robust signal when using brain material.

7. Please explain any problems you had with the antibody and/or experimental results that Aviva can address in the future.

We were not able to detect endogenous UCHL1 on rat liver cytosol and HeLa cells. We believe that in these protein extracts, UCHL1 is expressed at very low levels.