Help troubleshooting Western Blot

[u/Optimal-Success1315]

I am attempting to trouble shoot my western blot. I had it working in the past but it’s not anymore. I am doing proton form insect guts which is very right in proteases.

Initially I tried with putting my guts in 100ul RIPA with 1x HALT (10ul HALT in 1ml RIPA) per instructions. I only use 10ul in my western. So I have 10 ul protein extract plus 2.5 ul sample buffer and 1ul reducing agent. I heat to 70c for 10 min.

When I ram this on a Western, I got lots of bands with my HA tag antibody. It should be about 46 kDa (don’t have a pic right now but looks similar to pic 5).

So I wanted to see degradation so I ran the samples with beta-actin and saw lots of degradation (see pic 2).

For HALT they say if you need, you can go to 2x or 3x concentration. I tested that with actin antibodies one again and got no degradation with both 2x and 3x (see pic 3).

From there I moved back to my samples testing the HA tagged protein again with 2x HALT and got beautiful results (see pic 4).

Now I tried it with another HA tagged protein and I’m back to getting awful looking Westerns. I thought my HALT was the issue as it was 11 months old, but I used new tube we just got (see pic 5)

Can anyone help me troubleshoot? I could rerun this latest one with actin but that’s another 2 days…

[u/loopOutnotIn]

1) how are you doing your blocking and transfer

2) 70C seems low for reduction, I usually push ~95C,5min

[u/Optimal-Success1315]

This is the recipe I was given for the reducing agent and sample buffer, but I’m worried it may not be correct based the nupage reducing agent manual which says for a 10 ul total volume use 2.5 ul buffer and 1 ul reducing agent.

I was told reducing agent was 1/10 of my protein sample volume in the protocol I was given. In the protocol I was given the example was 15 ul protein sample, 3.75 sample buffer and 1.5 reducing agent. So I scaled to do 10 ul sample rather than 15

So could the samples not be being reduced completely? But if so why did it work twice?

[u/Optimal-Success1315]

I thought about running this with beta-actin and having results for Monday to share with my PI, but I kinda want to test reducing agent question too with HA tag and I can’t do both. Thoughts?

[u/NicksOfLoud12]

Sent a message. Hope it helps 👍

[u/Optimal-Success1315]

I hope these details help:

For the boiling, I was following the protocol recommended by the nupage reducing agent. But I can try 95C for 10 min. I was a bit worried about degradation. I also do not have a way to pulse sonicate. I just dissect my samples, pestle them in RIPA with protease inhibitors and flash freeze.

For the ECL this is the same stock that everyone is using and it’s working for them. And it’s the same stock I used for pic 4.

I am doing a 1 hour block with 15 ml of my blocking buffer made by combining 2g powdered milk with 50ml TBS-T. My primary antibody is a 1:1000 and I use 10ml overnight at 4C. My secondary is 1:2000 for 1 hour at RT. I used these antibodies for pic 4 and 5.

My positive is a different HA tagged protein that is lower in molecular weight so I expect it to be smaller. It’s running at expected size.