Hello everyone, I am trying to visualise ABCA1 protein (which is approximately 220-240 KD) by western blot from my experimental in-vitro cell samples. To extract proteins, I use either: NucleoSpin RNA/Protein extraction kit (Machery Nagel), or traditional cell lysis buffer or RIPA buffer. I can clearly see strong and clear bands for the house-keeping gene (actin), but the ABCA1 seems either, very faint, smear like or blurry. I am using ABCAM ABCA1 antibody, and loading the appropriate protein quantity as per the antibody manufacturer instructions. I am using either 1.5 or 1.0mm gel and bio-rad tetra system to do the run. Could anyone suggest how can i improve the abca1 band visualization ?. Thanks in advance.
Samrat Das you can try optimising the following components:
1. By increasing protein loading concentration
2. Increasing the antibody concentration up to 1: 100 ( For instance; 1uL antibody in 100uL Skim milk or BSA)
3. Increase transfer period (100 V for 3hours)
4. Use 6% SDS gel
In addition, you can compare antibodies manufactured by different companies.
Samrat Das since your protein of interest is around 220-240 KDa, I suggest the following steps.
1. Use a low percentage gel about 7.5%
2. Optimize the transfer conditions. It's better to use wet transfer to visualize high molecular weight proteins. If you're uing the semi-dry method, transfer at 20V for 1 hour.
3. Increase the protein concentration.
What kind of gel are you using? You might want to consider Tris-acetate gels, if you are not using that already, as they are specifically for resolving high MW proteins.
I forgot to add, for large proteins, I strongly recommend doing and overnight wet transfer (11 mA). Large proteins don't transfer efficiently so they need extra time. It's very common to not even see high MW proteins on a WB after a 2 hour transfer.
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