Anti-Topoisomerase I antibody [Mab1] (ab12407)

ORDER NUMBER 55951

DESCRIPTION OF THE PROBLEM Multiple bands, I have a band at~100kDa but also at~30-40kDa, this band only showed in my treated sample

SAMPLE MRC-5 cell lysis (human fibroblast cell-line)

PRIMARY ANTIBODY diluted primary antibody in 5%milk/TBST 1:1000, inc. overnight on rotator at 4 degrees

SECONDARY ANTIBODY washed 4 times for 10mins each wash and fresh buffer before and after secondary antibody.

diluted in 5%milk/TBST at 1:2000, anti-rabbit HRP, inc. 1hour RT

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED molecular weight marker (magic mark and see blue plus

ran a fibrillarin gel at same time and worked great.

neg control showed slight band at ~100kDA

ANTIBODY STORAGE CONDITIONS stored in aliquotts of 5ul in -80

SAMPLE PREPARATION used lysis buffer with NaCl,MgCl2,Trizma base and triton, at time of lysis added 1mM PMSF and a protease inhibitor tablet, sat 5min, syringe expelled lysate numerous times through increasingly smaller gauge needles

AMOUNT OF PROTEIN LOADED 20ug protien

ELECTROPHORESIS/GEL CONDITIONS Used invitrogen NuPage gel system as per manufactures protocol. MOPS running buffer on a 4-12% bis-tris gel

TRANSFER AND BLOCKING CONDITIONS transfered using Biorad transfer rig and instructions, blocked for an hour at RT in 5% milk/TBST

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? primary incubation, secondary dilution and incubation

ADDITIONAL NOTES does this antibody have a cleaved portion? it is know to have a cleaved portion at ~70kDa called Scl-70, however this band is too small.

ANSWER:

Thank you for your enquiry and for providing more details of your protocol.

This antibody has been tested for Western blot application using A231 human kidney cells. Thus, we would suggest running a positive control along with your samples.

As we understand from your e-mail, using MRC-5 cell lysis (human fibroblast cell-line) you can see the main band at 100 kDa on the membrane but also see another band at 30-40kDa only in treated cells. Is that right? Unfortunately, we do not know what that band represents but it is interesting that it appears only in treated cells.

You have not mention which treatment or drug you used. It would be interesting to know and check the viability of the cells after treatment.

We would perhaps suggest lowering the concentration of the primary antibody and see if the non-specific bands can still be seen.

We hope that this will help.