Hello,

I am troubleshooting a western protocol using whole heart homogenates but I seem to be getting consistent smearing at the lower band range of my gel which is also where I want to see my loading control band for b-actin ~42kDa [mouse monoclonal]. See image below.

Coomassie stain of the separation showed distinct bands in the higher molecular range [faint] but the smearing is present in the lower band range. Transfer success I have not checked with Ponceau staining yet [just the ladder transfer check]

I have made some adjustments to the protocol but I am mostly attributing the issue to poor sample preparation. Either because 1) degradation of protein 2) Too much protein 3) poor lysing

Would someone share their sample preparation protocol for their whole heart lysis?

I have included my most recent version of protocol associated with the image.

Summarized Sample preparation Protocol

1. Heart was retrieved from -80C [I have also done fresh heart collection but the smearing persists]

2. Add ~100ul of cold RIPA buffer with protease inhibitor for every 10mg of tissue. [I have used so far 500ul RIPA for ~70mg of heart tissue]

3. While tube in ice, use tissue homogenizer in small pulses until no solid tissue.

4. Let the homogenate sit 30mins in 4C ice with intervals of vortexing.

5. Centrifuge at 14 000 RPM at 4C for 10min to pellet cell debris, and then transfer the supernatant to a fresh microfuge tube. Repeat until clear lysate

Protein Electrophoresis Separation

1. Determine protein concentration of the lysate by BCA protein assay and dilute to ~20ug/well

2. All protein samples were mixed with 20ul 2x SDS loading buffer + 4ul reducing agent and heated at 85C for 2 min [as recommended, previously I had done 93C for 5min]> vortex>let cool

3. ~20 ug of total protein was loaded onto wells in 8% SDS-polyacrylamide gel.

Transfer

1. Using PVDF primed in methanol and transfer buffer with 20% methanol

2. Run 20V for ~45 mins

Primary & Secondary Antibody Incubation

1. Incubate with with 5% BSA or dry milk in TBS for blocking

2. Transfer membranes were incubated with primary antibodies at 4C overnight

3. Washed with TBST, 3 times-five minutes each wash

4. Incubate with HRP conjugated secondary antibody for 1 h at RT with gentle agitation

5. Wash 5 times, five minutes each wash with TBST

a. Signal Detection

6. Signals were detected by employing ECL kit

I would appreciate any insight!

Thank you