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Preparing enough sample for multiple westerns

Preparing enough sample for multiple westerns - Western Blot Forum

Preparing enough sample for multiple westerns - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 06-20-2012, 01:18 PM
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Default Preparing enough sample for multiple westerns



In the past I have always prepared enough sample for one western blot.

For example:
Sample A = 3.33ug/uL
Need 40ug;
Therefore:
12uL of Sample A
+ 12uL of 2x Laemmli
+ 10uL of H20
------------------------
34uL of Sample A for western
Boil 8 mins
Load 34uL

However, I'm getting tired of doing this for each western and would like to only do it once. So would this work?

Sample A = 3.33ug/ul
Need 40ug per western;
Therefore for 5 westerns:
60uL of Sample A (5 x 12ul)
+60uL of 2x Laemmli
+10uL of H20
---------------------------------
130uL of Sample A for 5 westerns
Boil 8 mins
Load 26uL


Would this work and are these calcs correct (would 26uL = 40ug)? Thanks!

Last edited by jeeep; 06-20-2012 at 01:19 PM.
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  #2  
Old 06-21-2012, 04:28 AM
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Default Re: Preparing enough sample for multiple westerns

looks good but why addition of H2O?
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Old 06-21-2012, 06:03 PM
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Default Re: Preparing enough sample for multiple westerns

All of my samples vary in protein concentration. The H2O brings everything to a consistent amount, so that when I'm loading I can load the same amount for each sample.

I should have the results later today.
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Old 06-22-2012, 05:04 AM
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Default Re: Preparing enough sample for multiple westerns

Do update us
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Old 06-23-2012, 10:44 PM
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Default Re: Preparing enough sample for multiple westerns



beta-actin look fine so it works
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Old 06-25-2012, 05:51 AM
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Default Re: Preparing enough sample for multiple westerns

wait you didn't use preparative gel? Cropped gel profile look good, some light smear. But is it just me or 2nd and 5th lane looked slightly darker.

With preparative gel it look pretty much more even since only 1 lane
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Old 06-25-2012, 08:34 AM
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Default Re: Preparing enough sample for multiple westerns

Sorry, but what do you mean by "preparative gel"? Also, it's not cropped. I cut this membrane twice and probed for proteins at 80, 44 (actin) and 17 kDa (saves time). There is some actin variation between samples but I can't seem to eliminate this. Any suggestions? Would you like my protocol?
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Old 06-25-2012, 03:50 PM
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Default Re: Preparing enough sample for multiple westerns

Sorry, but what do you mean by "preparative gel"? Also, the membrane was cut in two place to expedite the whole process. That way I can probe for a protein at 75kDa, 44kDa (actin) and 17kDa.

I never can seem to get perfect actins. Any idea?
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Old 06-26-2012, 06:41 AM
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Default Re: Preparing enough sample for multiple westerns

ops maybe my bad, you are doing multiple sample for one western blot and one gel is it?

Preparative gel where a comb with only 2 or 3 wells, loading the ladder on the smaller well and single sample type on the other well. Since it is only one well thus the homogenicity would be even.
something like
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