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Western Blot-No Sample Band, but Positive Control band visible

Western Blot Homogenization; Protein Degradation; No Bands; Beta-actin;

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#1 niz

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Posted 23 July 2013 - 07:59 PM

Hello,
I was trying to do Western Blot to detect beta-actin in mouse liver homogenite.
I am using nitro-cellulose membrane, and ECL for detection.
I have commercial mouse liver homogenite which I am using as positive control.

I am getting good bands at ~40KDa for the positive control, but no band for the samples.
I loaded 20ug- 50ug of protein per well.

I do not know what the problem is, but it seems like I have problem with my sample preparation.
Here is how I prepared the samples-
The livers were collected and flash frozen, and then kept at -80 C.
I homogenized ~40mg- 50mg of each liver in the following 1ml of ice-cold buffer (modified RIPA buffer).
Tris HCl, pH 7.5 --- 50mM
NaCl------------------ 0.5 M
EDTA----------------- 2mM
EGTA ---------------- 2mM
TritonX-100----------- 1%
Sodium Deoxycholate-- 0.25%

Just before the experiment, following protease inhibitors were added-
NaF---------------------- 1mM
Na3VO4(activated)---- 1mM
DTT --------------------- 2mM
Protease inhibitor cocktail Set 1 (emd millipore) ---- 10ul per 1ml of buffer

I used Dounce glass rods to homogenize the samples (10-12 strokes in ice).
Then the homogenate was kept in ice for 30 mins for the surfactants to work.
Then it was centrifuged @ 15,000 rpm for 15 mins & the middle supernatant was collected & aliquoted in -80 C.

I did the protein assay, and can quantify the proteins. The concentrations is about 15ug/ ul,
but still no beta-actin bands. I repeated the experiment 3 times, and got the same results with No sample band, but positive bands only.


I am not sure what can be wrong. Please help me!!!

Edited by niz, 23 July 2013 - 08:03 PM.


#2 bob1

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Posted 24 July 2013 - 01:16 AM

Livers are absolutely full of proteases, try adding twice what you have there.

#3 mdfenko

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Posted 24 July 2013 - 04:37 AM

actin will polymerize at high salt. 0.5M in the extraction medium may be your problem. check the pellet for actin.


by the way, fluoride and vanadate are phosphatase inhibitors, dtt is a reducing agent.
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#4 niz

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Posted 24 July 2013 - 07:56 AM

Thank you guys for your replies. I will reduce the NaCl conc. from 0.5M to 150mM and will rehomoginate the samples.

And I will also try doubling the protease inhibitor conc.


Here is what I have in my protease inhibitor cocktail:

Protease Inhibitor Name

1X Conc.


AEBSF

500uM


Aprotinin

150nm


E-64 protease inhibitor

1uM


EDTA

0.5mM


Leupeptin

1uM



Do you think I should try another cocktail set? or this one is sufficient?

Also, should I add 1mM of PMSF in the buffer as well?

@mdfenko..................my bad

#5 mdfenko

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Posted 25 July 2013 - 04:10 AM

that cocktail should be sufficient. aebsf is a less toxic, more stable alternative to pmsf.

Edited by mdfenko, 25 July 2013 - 04:10 AM.

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genius does what it must
i do what i get paid to do

#6 niz

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Posted 26 July 2013 - 07:46 AM

Thanks again. I will run the gel over the weekend and will post the results.

#7 rkay447

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Posted 02 August 2013 - 11:14 AM

I am wondering if you ponceau your membrane after transfer?  If so, do you see good strong bands of protein in your samples?  I also agree that half molar salt could precipitate proteins.  Hope you remember to update.



#8 niz

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Posted 07 August 2013 - 12:02 PM

I rehomogenize the samples using new buffer having 150mM NaCl but no luck so far.

 

I did not do ponceau, actually I am new to western blotting and never did the ponceau staining. Next time I will do.

 

Do you think I should reduce the NaCl conc. to around 100mM? 

 

Can anyone tell me the purpose of NaCl in RIPA buffer? and if I want to prepare whole cell extract from mouse liver, do I NaCl?

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Edited by niz, 07 August 2013 - 12:05 PM.


#9 bob1

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Posted 07 August 2013 - 12:40 PM

NaCl is used as osmolarity level, 150 mM is approximately the concentration of salts in the body and as such is the "normal" condition for proteins to exist in, so it doesn't damage or change folding and similar things.


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#10 rkay447

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Posted 07 August 2013 - 02:21 PM

Agree with bob1.  Salt is important for maintaining a normal physiological condition that supports protein structure, binding, stability, ect.  It's important to have at least some in your buffer.  I use a buffer with 50mM with no problems but I've also used 150mM.  You can take the membrane you posted and ponceau it to see if your samples actually have the amount of protein you are expecting.  How are you quantifying the amount of protein in your sample?  I assume bradford but never hurts to ask.  Please ponceau your membrane and let us know or show us how it looks.



#11 mdfenko

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Posted 08 August 2013 - 01:15 PM

actin will polymerize in relatively low concentrations of salt. we routinely polymerize in 100mM and can get it to polymerize in 50mM.

 

you may be pelleting the actin before you take the supernate for the gel.

 

have you checked the pellet for actin?


talent does what it can
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i do what i get paid to do





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