western blot q

[chef]

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hey western gurus -
is it possible to reuse a blot after western blotting? it's a nitrocellulose membrane and I used a mouse ab/anti-mouse HRP secondary w/o much success.. so i want to try a rabbit primary ab that gives a band of the same size. thanks!

 

[dr2bjake]

hey western gurus -
is it possible to reuse a blot after western blotting? it's a nitrocellulose membrane and I used a mouse ab/anti-mouse HRP secondary w/o much success.. so i want to try a rabbit primary ab that gives a band of the same size. thanks!

Absolutely, once you are finished the transfer step you CAN re-use the nitrocellulose membrane.

[For storage just dry it out, put it inside some filter paper, and store in saran wrap] = good for a long time.

Wash with TBST multiple times (I do atleast 5 washes) then add stripping buffer (commercially available). I incubate in stripping buffer for about 30-45 minutes in a water bath equilibrated to room temperature.

Although, if its the same molecular weight you are after and just got no signal, then I would just ignore the Stripping Buffer step. Its not necessary. Adding stripping buffer actually degrades some of the protein left on the membrane.

Other questions to conisder are:
1) Did you load enough protein (ug sample)?
2) Are you using a NEW ECL detection kit for the HRP labelled secondary
(sometimes a bad kit will mess everything up)?
3) Did you block with Milk?
4) And this is the obvious one... did you see the protein ladder transfer onto the membrane?

Hope this answers your question!

Good luck!

I hate westerns...

 

[Doctor&Geek]

You can make your own stripping buffer easily with beta-mercaptoethanol, 10% SDS, Tris pH 6.8, and water.

375 uL BME
10mL 10% SDS
3.25mL Tris pH 6.8
Water up to 50mL

Put into tray along with blot, cover with saran wrap, and put into 50 degree rotating water bath for 30 minutes.

Wash a few times with ddH20, then do 2 TTBS washes, 10 minutes each. Block, then reprobe with primary.

You will lose some proteins each time you strip, so do it sparingly, and don't let the stripping step last too long.

 

[xanthines]

I also incubate with ECL or whatever detection agent before re-probing to make sure that the first set of Ab's were completely stripped. I do this especially when reprobing for different proteins of the same size. You don't want any false signals!

-X

You can make your own stripping buffer easily with beta-mercaptoethanol, 10% SDS, Tris pH 6.8, and water.

375 uL BME
10mL 10% SDS
3.25mL Tris pH 6.8
Water up to 50mL

Put into tray along with blot, cover with saran wrap, and put into 50 degree rotating water bath for 30 minutes.

Wash a few times with ddH20, then do 2 TTBS washes, 10 minutes each. Block, then reprobe with primary.

You will lose some proteins each time you strip, so do it sparingly, and don't let the stripping step last too long.

 

[dr.z]

If you want to re-use membranes, I recommend PVDF which is more durable. Stripping and re-using nitrocellulose may get messy after a while.

 

[linuxizer]

Wash with TBST multiple times (I do atleast 5 washes) then add stripping buffer (commercially available). I incubate in stripping buffer for about 30-45 minutes in a water bath equilibrated to room temperature.
I hate westerns...

You forgot the most important step, inserted like this:
1. Wash with TBST multiple times.
2. Hold nose.
3. Add stripping buffer as quickly as possible so that the your labmates don't hate you.

I hate that stuff!

--Ari

 

[b&ierstiefel]

You forgot the most important step, inserted like this:
1. Wash with TBST multiple times.
2. Hold nose.
3. Add stripping buffer as quickly as possible so that the your labmates don't hate you.

I hate that stuff!

--Ari

The worst isn't when people add the stripping buffer to the membrane. It's AFTER the stripping when they pour that crap down the sink and don't flush it down with running water. Now that stinks! That stuff smells like turd covered with burnt hair.

 

[Doctor&Geek]

Don't forget the disposable tips you use to dispense BME. Those stink up too.

 

[linuxizer]

Don't forget the disposable tips you use to dispense BME. Those stink up too.

Yeah, and the sink problem is bad too. However, the rotting fish smell of TEMED has few equals, it's just that no one pours 30mL of TEMED down the drain like they do stripping buffer. Oh, and I *hate* it when the vacuum traps get emptied and someone forgot to thoroughly bleach the bottom. The whole floor smells like bacteria for the day.
Sincerely,
Your friendly neighborhood pre-MSTP with an overly-sensitive nose

 

[b&ierstiefel]

If there's one thing I've learned while working in a lab setting all these years is that labs can be filled with irresponsible, inconsiderate ******s. Don't get me wrong, they're good people. But they just don't connect the dots and think, "how could this affect the others around me?"

These people will dump their used stripping solution (or God forbid, TEMED...never seen that poured) down the sinks and not flush the sink with water to minimize the pungent odor. Instead, they walk all the way to their lab benches where they won't be affected by the smell and think, "it's all good." I wonder sometimes what their home lives are like...if their significant others, spouses, or roommates/housemates tolerate this kind of crap around the house/apartment. Kinda like sh*ttin' on the throne and not flushing.

The other lab mentality that really annoys the f*ck outta me is the "if I don't do it, somebody else will get pissed off enough to do it for me...hence, I don't have to do it!" This especially applies to situations where somebody uses up a reagent or materials and "conveniently forgets" to re-order it thereby screwing the next person who needs it and will therefore order it.

 

[linuxizer]

The other lab mentality that really annoys the f*ck outta me is the "if I don't do it, somebody else will get pissed off enough to do it for me...hence, I don't have to do it!" This especially applies to situations where somebody uses up a reagent or materials and "conveniently forgets" to re-order it thereby screwing the next person who needs it and will therefore order it.

Yeah, and the "I'm not going to learn where things go I'll just put them wherever I want" mentality, and the "I'm going to get grumpy when you ask to borrow 5uL of a cheap solution that I have 500L of, despite having borrowed half your similar solution last week" mentality, and the "I'm going to leave all the lab's markers point up so they never write well" mentality.
Ok, maybe that last one's just my pet peeve 😛.
Ari