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caspase3 detection


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#1 xuezhan6381

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Posted 13 May 2009 - 09:01 AM

i detect cleaved-caspase3 with 15% sepreating gel(1.5mm), tank transfer at 110V for 90min, 0.2um NC membrane, loading 40-50ul sample/lane(sample: 4X106cell lysis in 240ul lysis buffer, add 80ul 4Xloadng buffer) .but the stained membrane showed diffuse and weak band at 15-20kd, the ECL result also weak.
how to improve my procedure to get a better result? please help me.
i use a549 cell, and the apoptosis is obvious.
the pre-caspase3 can be clearly detected.

#2 dtimm

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Posted 13 May 2009 - 02:11 PM

Adding 15-20% MeOH in your transfer buffer will help transfer low MW proteins. Stain the filter paper behind your membrane to see if there is any blowthrough. If so, reduce your voltage (ex. 80v for 1 hour 20 mins.).

#3 xuezhan6381

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Posted 13 May 2009 - 04:44 PM

Adding 15-20% MeOH in your transfer buffer will help transfer low MW proteins. Stain the filter paper behind your membrane to see if there is any blowthrough. If so, reduce your voltage (ex. 80v for 1 hour 20 mins.).

is it possible that protein between >20kd pass through 0.22um NC membrane ? i haven't stained the filter ,but i have check the reverse side of membrane ,there is no band

#4 bob1

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Posted 13 May 2009 - 05:51 PM

I would definitely transfer a lot slower - we use 30v for 2 hours. It is entirely possible that your protein has passed through the membrane. To get over this problem I suggest using PVDF rather than NC.

#5 xuezhan6381

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Posted 13 May 2009 - 08:22 PM

I would definitely transfer a lot slower - we use 30v for 2 hours. It is entirely possible that your protein has passed through the membrane. To get over this problem I suggest using PVDF rather than NC.

but many western manual point out 0.2um NC prevent small pretein from passing through the membrane, and i can see 15kd marker clearly on the stained membrane . how to explain that

#6 Aris

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Posted 22 May 2009 - 06:36 PM

I would definitely transfer a lot slower - we use 30v for 2 hours. It is entirely possible that your protein has passed through the membrane. To get over this problem I suggest using PVDF rather than NC.

but many western manual point out 0.2um NC prevent small pretein from passing through the membrane, and i can see 15kd marker clearly on the stained membrane . how to explain that


To start with, there is no such thing as complete transfer. If you call up the company (Invitrogen??) and ask them the same question, they will definetely say to you that there is a blow through right from the very first time you start the trasfer. The point is how much blow through you have. NC membranes are desinged in that way so they are more charged and give much less background. But you cannot avoid the blow through. When facing this kind of problems when you have a protein in very small percentage in your sample (as Caspase usually) it would help sometimes to change to PVDF since PVDFs tend to bind proteins stronger than NC, thus occasionally more background.
Now because I have worked with Caspase, this is a small protein. So the smaller the pores of the blot, the better your results. The NC that you have is ok but PVDFs with larger pores (0,4 for example) will also do. I have done this for even smaller proteins than Caspase.
The amount you are loading is ok. I would also go for 50ug per well and I have also done it in the past using PVDF membranes. Worked just fine.
Now, you are doing wet transfer i guess. I have never heard of such Voltage adjustments. Try reducing to 30 overnight or 50 for 3 hours. If you are doing semidry, then 20Volts for 45 minutes will do. Even with even 15 minutes you will see results on the blot, but only for the small proteins.
Regarding to the molecular weight markers, these are just markers. They dont necessarily behave like the proteins. Sometimes you will see them on the other side of the blot, sometimes not depending on the timeframe you run the transfer.
Stain your blot right after the transfer to see where you have what. If you do this after the blocking part and after incubating then the bands will not be as good as before blocking. Then destain your blot with TBST and continue with blocking and so on.
For me the primary from Cell signaling worked just fine. The secondy anti-rabbit worked also very nice. Somtimes the anti-goat Abs are problematic but anti-rabbit more or less ok.
Keep in touch.
Cheers.




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