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
Dear Janna,
You should try gels with higher agarose percentages around 10%-12% to resolve your 40 kDa protein. I think this is your biggest issue. I recommend MOPs gels.
You may also have too much protein. Regulate your protein lysate to a final concentration of 2 ug/ul (including the complete loading buffer) and boil 10 minutes at 95 C.
Cheers,
Hello Sebastián,
As you suggested, I have recently diluted my samples and I have an improved separation and distinct bands based on coomassie. But funny story, I now see no bands after blotting! This is after restarting with fresh samples/solutions. I am not sure if this is related to my original issue with my protocol but I hope to come to a happy medium.
I may also consider using 10% as you suggested.
Thank you for your response.
Keep trying, blots normally tend to get funny near end of year!
Good luck
First, an 8% gel should be fine, but think about the buffer (bis-tris is better for low molecular weight resolution than tris-acetate). Lysis buffer volume to tissue mass for me was about 20mgs powdered tissue in about 350µL lysis buffer with inhibitors.
I had a similar problem with mouse heart tissue. It turned out that heart has a different kind of actin than other tissues. You need to use a heart specific antibody (e.g. Cell Signaling #8456 ). I did my protein isolation using Pierce TPER with HALT protease/phosphatase inhibitor solution added to 1X final. I got very nice banding when I ran 40µg protein per lane, and (importantly) ran my gel for 1 hour at 80 volts before taking it up to 125 or 150 volts. It makes a big difference in the appearance of the banding on a Western with tissue lysates.
- Badges
- Science method