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Western problem - 8 months now...(long)


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#1 Mummytomax

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Posted 12 January 2005 - 02:44 PM

Hi, just found this site this morning, hopefully one of you can shed some light on my problem....

Iím having problems with a western blot (oh shock horror! :o ) and no suggestion from the companies have worked and each has said itís the otherís antibodies problem and I thinks itís something else entirely (to which no one Iíve asked in RL has been able to solve).

I'm measuring the effect of 2 drugs on AKT and pAKT(ser473 and thr308) expression in human prostate carcinoma cell line DU145 with wortmannin and EGF and my negative and positive controls.

I was getting 'funny' results (every blot looked the same, no difference was seen with treatment or antibody) so after eliminating possibilities with Cell Signaling (the manufactures of the primary) I probed with the secondary only and got 'beautiful' films with banding exactly at 60kDa - the point where I wanted to visualize my Akt and pAkt.

A coomassie of the gel shows a large dark protein band occurs at 60 kDa in all samples. Does anyone have any idea what this could be and if it is masking my results by unspecific binding with the secondary how I could get around it?

The problem is occurring with both the anti-mouse and the anti-rabbit Vectastain Elite ABC kits PK 6101 and PK 6102.I've tried diluting the secondary further but am now down to 1:50,000 and still have a HUGE dark band in every lane. Would be wonderful picture if the results were real.

Following treatment the cells are scraped and lysed on ice with 20mM Tris (pH 7.5), 150 mM NaCl, 1mM EGTA, 1mM EDTA, 1% triton, 1 % glycerol, 1 mM sodium orthophsphate, 1 ug/mL leupetin, 10 ug/mL Apropotinin, and 1 mM PMSF to inhibit dephosphorylation of my proteins of interest. Sample buffer is simple Lamaeli buffer.

I've tried varying my blocking from 2-10% skim milk, BSA, horse serum, goat serum, human serum, FCS, FBS in TBBS with tween 20.and I'm at a loss as to what to do next. And blocking times Iíve varied between 2 hours and overnight. Visualization is 30 secs with ECL.

I plan to try a secondary from a different company next week and see if it still shows the false banding. Any other ideas gladly welcomed!!!!

#2 ajames

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Posted 12 January 2005 - 09:23 PM

BSA has a molecular weight at approximately this point. The tissue culture media you are growing your cells in probably contains some form of calf serum which is very high in BSA. Also many antigen preps are "contaminated" with BSA so if your secondary antibody is polyclonal it may contain antibodies that react with BSA. All I can suggest is that you wash your cells with lots of ice cold PBS before extracing protein. If your cells are not washed sufficiently the majority of protein you are loading may actually be drived from the calf serum and not the cells of interest since it is already nicely in solution even before you add the lysis buffer.

#3 Mummytomax

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Posted 13 January 2005 - 01:45 PM

Duh! That's so obvious.

I was thinking it could be HSA from the cells themselves as it also is 66 kDa and was looking at a cartridge to remove it. Washing the cells more is a much simple thing to try! B)




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