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western blot - problems with detection - (Sep/01/2005 )

Hi,

I am trying to see C-Raf & Cdk4 in PC3 cell lysates using western blot. I use PVDF membrane and BioRad protocol. My primary antibodies are from Santa Cruz. I use IR dye conjugated secondary anti-rabbit (Rockland) and detect using Odyssey scanner and use their protocol after transfer (i use the Odyssey blocking buffer). I tried several different dilutions of primaries and sec.anti body & different protein loads (maximum 70 microg per lane) but still couldn't see clearly visible bands.

I prepare the cell lysates using the following method: Trypsinize cells (500 microl of trypsin + 500 microl of medium), then spun to collect the pellets. Stored the pellets at -80degrees overnight for complete lysis. Then added 50 microl of cell lysis buffer (containing Hepes buffer, CHAPS, Nacl, sodium orthovanadate, NaF, 2-glycerophosphate, EDTA, DTT, PC cocktail and PMSF). Incubate for 30 min at 4 degree C. Then spin at 12000 rpm for 10 min to collect supernatants for loading.

Please give your valuable suggestions.

Thank you!

-smwestern-

hi
i think there can be several reasons for this.........
1st check if your monoclonal is too old coz this had happened to a friend of mine who was supplied with an old batch of monoclonals
check if the monoclonal reacts witha denatured or native form of the antigen. n process your samples accordingly
check that your washing steps are not too stringent, blocking not too much n transfer is complete
hope this helped
best of luck!

-scorpaps-

QUOTE
My primary antibodies are from Santa Cruz



There is your problem. Antibodies from Santa Cruz are: (pardon my language) shite

-pBluescript-

QUOTE (pBluescript @ Sep 6 2005, 06:01 PM)
QUOTE
My primary antibodies are from Santa Cruz



There is your problem. Antibodies from Santa Cruz are: (pardon my language) shite




Hello!

All of my primary antibodies are purchased by Santa cruz. If they are bad. do you have any good manufacturers? I can not detect my actin and some other antibodies. ohmy.gif

Thanks alot!

-calmdown-

For detection with the Odyssey system, Nitrocellulose membranes give much less background. Also, non fat dry milk (3%) is better than their blocking solution.

DO NOT incubate with your 2nd Ab for more than 1h.

DO NOT use Tween in your washing solutions.

Hope it helps

-UniSPheryk-

QUOTE (UniSPheryk @ Sep 29 2005, 07:19 AM)
For detection with the Odyssey system, Nitrocellulose membranes give much less background. Also, non fat dry milk (3%) is better than their blocking solution.

DO NOT incubate with your 2nd Ab for more than 1h.

DO NOT use Tween in your washing solutions.

Hope it helps

could you tell me why not to use Tween in the washing solutions or what's role of Tween-20 in solutions?

-biolove-

QUOTE (biolove @ Sep 29 2005, 06:09 PM)
QUOTE (UniSPheryk @ Sep 29 2005, 07:19 AM)

For detection with the Odyssey system, Nitrocellulose membranes give much less background. Also, non fat dry milk (3%) is better than their blocking solution.

DO NOT incubate with your 2nd Ab for more than 1h.

DO NOT use Tween in your washing solutions.

Hope it helps

could you tell me why not to use Tween in the washing solutions or what's role of Tween-20 in solutions?


As far as I know, Tween in washing solutions (or blocking solution) avoid cross-reactivity and background. I'm surprised with this advice, too, but perhaps too mush washes or aggressive washes is better in your case. blink.gif

-E.G-

[q]There is your problem. Antibodies from Santa Cruz are: (pardon my language) shite[/q]

I totally agree with this!!

And about tween you should use it in your wash, when I don't my background rises!

hope it helps!

-SMH-