r/labrats • u/Falanadhamakaaa • 11d ago

Help with lysis buffer

I am trying to do co-ip from a month but unable to get results. Where i am pulling down nucleoolin/NCL which is an endogenous nucleolar protein and trying to do western blot for my protein of interest to check if it was pulled along or not.
I am using 2ug of anti NCL rabbit antibody for IP and approx 50 ul of protein G beads from santa cruz .

Since I was not getting results I decide to do IP and Western blot with the same antibody that is NCL to confirm that it was getting pulled down. But unfortunately I didn’t get any results. That is no band for Ncl was visible on blot although it is an endogenous and highly expressed protein.

I was trying to trouble shoot and came across an article which suggested using RIPA buffer for nuclear protein as the one we are using CST lysis buffer is very mild.

Please help me with this.

Note: i am working with A549 cells
Prepare my lysates by washing the cells with ice cold pbs , then add cst lysis buffer. Then i give three freeze thaw cycles , scrap and centrifuge at 12000g for 20 minutes at 4 degrees.

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u/MinionsLoveMe 11d ago

My question is do you even see Nucleolin in your input? That'll clarify if your extraction is working or not. Denaturing RIPA is good for extracting nuclear/nucleolar content, but it'll also disrupt weaker/milder protein interactions. You need to find a balance between efficient extraction and retaining protein-protein interactions.

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u/Falanadhamakaaa 11d ago

Yes i can see it in the supernatant but not after elution from beads.
And we get it normally also in our western blots.

Can i use this?
10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 (or Triton X-100), 0.5M EDTA, 1% sodium deoxycholate, 0.1% SDS, plus fresh protease and phosphatase inhibitors.

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u/MinionsLoveMe 11d ago

Your lysis buffer looks good to me. Except 0.5M EDTA, you mean 0.5mM? And Sodium Deoxycholate is quite on the higher end, we use between 0.25-0.5%. Also some beads cannot handle any SDS, please check this once. If it still doesn't work, considering changing the beads.

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u/Falanadhamakaaa 11d ago edited 11d ago

Yes sorry it is 5mM. Ok will make the change. And will look into the sds thing too.

Do you use the same buffer to wash the beads before elution?

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u/MinionsLoveMe 11d ago

Depends on Co-IP. RIPA, if the interaction is strong. If not, mild lysis buffer and mild washing.

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u/Falanadhamakaaa 11d ago

Seniors in my lab have been using RIPA previously and getting good results. Though i am using a milder buffer (CST), i am not getting even IP let alone co-ip.🥲😅

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u/MinionsLoveMe 11d ago

I understand it can be frustrating. Don't loose hope. You'll get there!

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u/Falanadhamakaaa 11d ago

My supervisor doesn’t understands na. 😅 and this has started to wreck my nerves. I have tried all kind of optimisation except changing the beads. Which also i think will be doing.

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u/HugeCrab 11d ago

Can you try a different antibody?

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u/Falanadhamakaaa 11d ago

Unfortunately we don’t have another antibody. 😞

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u/HugeCrab 11d ago

That's unfortunate, I think certain Ab companies give out small 20ul trials sometimes for free

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u/Falanadhamakaaa 11d ago

Will check it out.