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Removing Albumin from Samples

Removing Albumin from Samples - Western Blot Forum

Removing Albumin from Samples - 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 01-03-2012, 08:05 PM
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Default Removing Albumin from Samples



Hey guys, I'm trying to analyze media that I've collected from growing cells for fluctuating levels of my protein of interest. Unfortunately, when I run my Western Blots, I get this fat streak/band of albumin which obscures any band I would possible be able to see. I was wondering if anyone has any ideas on the best way to remove albumin from my samples so that I can get clean WB results.
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Old 01-04-2012, 12:48 PM
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Default Re: Removing Albumin from Samples

i donot think there is any protocol to remove albumin from your sample. Here few suggestions
1. First try to optimise your antibody so that you can get band of your protein of interest. by getting good antibody or optimising its concentration etc.

2, Reduce serum concentration in your medium or if possible without serum.

3.if nothing is possible one wired idea is pull out albumin from your sample by immunoprecipitation.
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Old 01-04-2012, 10:16 PM
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Default Re: Removing Albumin from Samples

Ah, are you sure? I talked to my professor and he mentioned that there is several protocols that exist but I have had trouble finding them on PubMed. I know that they're are albumin kits available that are suppose to remove albumin from your sample but I need to do more research on that.
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Old 01-05-2012, 04:50 PM
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Default Re: Removing Albumin from Samples

Albumin can be removed from samples using a resin called Cibrachon Blue. There are several commercially available resins on the market, including AlbuminOUT from G-Biosciences.
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Old 01-06-2012, 12:39 AM
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Default Re: Removing Albumin from Samples

ColinH do you know any brand or method is more economical/cheapest yet effective?
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Old 01-06-2012, 03:46 PM
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Default Re: Removing Albumin from Samples

Affordable and effective, try the GBiosciences brand. They also have a small 4 column size to test.
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Old 01-07-2012, 08:30 AM
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Default Re: Removing Albumin from Samples

Thank you so much for the help!!

Colinh, so i would be able to use the gbiosciences kit to remove albumin from the media i collected from my cells (I usually use 5% BCS)? Do you have any experience determining if using the kit messed with your samples in anyway besides removing albumin?
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Old 01-08-2012, 06:35 PM
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Default Re: Removing Albumin from Samples

Ah what glorious info!! I just did some searching myself and answered my own question. Thanks so much ColinH and everoyne else for the info!! Much appreciated!!!
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Old 02-23-2012, 11:22 AM
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Default Re: Removing Albumin from Samples

Electrophoresis method to albumin remove from mixtureA PAGE, in the absence of denaturing reagent, was performed using Bio-Rad Slab Gel Electrophoresis system according to the manufacturer's instructions. Slab gels consist of a 7.5 % acrylamide resolving gel and a two parts of 4.5 % stacking gel respectively : a) load gel and b) Blue sepharose CL-6B gel. On the total length of the gel 14.8 % is load gel, 11.11 % blue sepharose CL-6B gel, and 74.1 % belongs resolving gel. The stacking gel containing Blue Sepharose CL-6B (Pharmacia) is constructed on top of the resolving gel and is prepared dissolving, with gently mix, 0.09 g of Blue Sepharose CL-6B per ml of stacking gel. Without delay, the slab gel plate is filled with the gel mixture. To avoid sedimentation of the Sepharose, the volume of ammonium persulfate is adjusted to obtain gel polymerization within 2 min. After polymerization, the stacking gel mixture without Blue Sepharose CL-6B is poured on top and a comb is inserted immediately to construct sample wells. Samples are prepared mixing 5-10 Ál of serum o supernatant of culture with 40 Ál of Tris-HCl 10 mM at pH 6.8 containing 20 % glycerol and 0.002 % of bromphenol blue. Twenty microliters of this sample is loaded into a well and initially electrophoresed at a current of 20 mA. After the bromphenol blue dye front had moved about 1 cm into the resolving gel, the current is increased to 40 mA. When the dye front reached the bottom of the gel, electrophoresis is stopped, stain with Coomassie brilliant blue R250 for protein detection, or transfer blot proteins by means electroblotted (Bio-Rad) onto a nitrocellulose membrane (Bio-Rad) for Western blot assay without albumin interference.
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