there could be many reasons, viz protein conc, antibody, methodology in general. please try to use some positive control (drugs). Load at least 50ug of protein in each well and use a good antibody for PARP.

Antibody not working or check and add proper secondary Ab.

Try with 12% gel but it will not be the issue because Actin and PARP1 has almost same mol wt.

If Actin is blot found it means protein is conc is okay.

No problem with protein. even 20 ug protein is enough for PARP1.

try a longer period of incubation with your primary antibody and also check different dilution of this antibody (try less diluted just to check if the antibody works). And of course, look into the literature which antibody is currently used.

Good luck

This result could be due to several factors such as the % of your gel, transfer time, how old is your transfer buffer, primary and secondary antibodies and their concentrations and incubation times, the level of PARP in MD 231 and also the amount that you loaded.

You should get a positive results usually with around 20-30 ug of total proteins in a 10% gel with approximately 1 hr transferring (95-105 V) using a new transfer buffer. When you develop for PARP, most of the time you will see PARP around 115-116 kDa ( depending on the conductivity of your transfer buffer exact values may change little) and cleaved-PARP around higher 80s because of the less specificity. CST Rb PARP has worked better for me several times but it gives a band around 89 kDa too (cleaved PARP). This will work 1:2000 with primary (overnight at 4 'C) and 1:3000 with 2 'ry for 2 hrs at RT. Use a new ECL solution and try with an enhancer if you do not see bands. Abcam will be the best antibody, but very expensive.

Good luck with your experiment.

I have investigated the expression of PARP in IL6 cell line and used Urea Extraction reducing Buffer .