**EDIT: 30ug of Protein is loaded measured by microBCA
I've been trying to blot for beta-actin on cell culture protein lysates for weeks without success. The image below is the best I have gotten. It is loaded with ladder, sample 1, 2, 3 twice.
Our lysates are from Bone-marrow mononuclear cells lyzed in RIPA + protease inhibitors and stored at -80. Protein concentrations are read by Pierce MicroBCA.
SDSPAGE: We do SDS-PAGE under reducing conditions in a 4-20% Tris-Glycine mini-gel with the Thermo Mini gel boxes. I run them at 180V for 1h15m. I've attached an image I get after a comassie stain of the gel (Imperial Protein Stain)
Transfer: After washing in dH2O, I transfer onto a PVDF membrane with the iBlot2 dry transfer and easily see the bands on the blot.
Blotting: I then block with 5% BSA (fresh) in TBST for 1h RT, incubate in b-actin antibody (CST, 1:1000) overnight at 4c. I then wash with TBST and incubate with anti-rabbit HRP secondary (abcam, 1:3000) 1h at RT.
Detection: I read the blot by adding the Amersham ECL reagent from GE for 1 minute and capturing on LAS3000 darkbox with 5m exposure.
I have troubleshooted the protein determination assay, the electrophoresis time, transfer time, and secondary antibody. I've not yet tried a new primary or ECL reagent.
What is going wrong??
1. you have no enough protein (ho much you think to have loaded?)how many cells the lysate is coming from?
2. Is the Ab working fine?. Have you tried with other housekeeping (GAPDH, H3, HSP90, vinculin, b-actin).
From the photos and your short description I am quite sure that your antibody or your ECL or the setting of the reader that you are using to image your wbcn be the potential problem. Proteins seem low but not that much. Moreover, usually the B-Actin Ab can be seen from the moon even with very low amount of protein...What's the Ab? Are you sure that the ECL is not expired? Have you got a positive control to test Ab and ECL??
BEst,
Chris
@Michaela
Thank you for your reply :)
1. I loaded 30ug of protein, which is usually more than enough for my previous applications.
2. I will be trying GAPDH and HSP90 as alternative loading controls per your recommendation!
Update:
Below is a blot with the first 3 lanes my bone-marrow stem cell lysate, the last two are lysates from endothelial cell lines (RAEC + HUVEC). The top blot is stained for AKT, the one below for beta-actin. It is clear that there is something up with my BMSC lysates, because beta-actin and AKT are staining very well for these non-primary lysates.
From this result, combined with prior results, I now believe that either 1) there is something in the BMSC lysates that is interfering with the BCA assay and giving me a poor reading. 2) the lysates are not very reactive to the antibodies I'm using.
The BMSC are plated on vitronectin-coated 10cm dishes. They are washed twice with cold PBS, then scraped into a tube with PBS. They are then pelleted and lyzed with 1x RIPA + protease inhibs at 100ul / 10e6 cells and incubated on ice for 15 minutes with periodic vortexing. They are then spun at 4c for 30m ~16000xg
Any new thoughts?
- Badges
- Science method