Can anyone tell me that for the isolation of chondrocytes from ear cartilage which type of collagenase is use?

Hi, I want to raise 2 questions and hope someone can help me to solve it.

(1) Everytime I do IP, no matter using protein a agarose (Calbiochem) or protein g sepharose fast flow (GE), I also found out that there are consistently a band about 55 kDa present on the blot.

I have excluded the possibility of IgG heavy and light chain contamination since I always use different species of antibody for IP and Western blot.

I encounter the same problem in different cell lines, and using different lysis buffers also can't solve the problem.

this problem only appears in the IP sample instead of the total sample.

(2) Also, I want to know after boiling the IP samples in sample buffer, can I just freeze the beads together with the samples in -20c without separating them?

Many thanks,

Tom

I've been doing EMSA's with FAM labeled DNA successfully but we recently switched over to an Odyssey detection system using IR Dye which is much more sensitive and I have run into a problem. I use a labeled primer to PCR up my labeled fragment, I use one labeled primer and one unlabeled. I now see two bands in my no protein lane. I think the upper band is single stranded DNA. I know it's not contamination because I have several different fragments with different primers and templates and its shows up in them all. To get rid of it I've increased the amount of the unlabeled primer and reduced the cycles in the PCR reaction. This has not worked, even with 15 fold unlabeled primer. Does anyone have any ideas? Thank you so much!

hello everybody
I'm doing a PhD in horse virology and it is more than 4 months that I can not develop an efficient protocol for Immunofluorescence of infected PBMC cells
I am using a monoclonal antibody that binds to a nucleocapsid protein. This product is available on the market
my problem is that this mAb binds aspecifically neutrophils (you can identify them for the morphology of the nucleus ... the really strange thing is that using an irrelevant isotype autofluorescence does not develop!
I tried to use a protocol to limit autofluorescence but was not successful
I do not know what to do, time passes and I always stop at the same point

I hope you can help

thanks

Dear Thomas,



The best, ideal and perfect way to abolish this problem is to fumigate your
4 degree/cold room. There are different ways to fumigate the best way is
KMnO4 (Potassium permanganate) + Formaldehyde (the amount of addition will
depend on size of your room) just i am illustrating the protocol



1. Vacate your room completely (the penetration and disinfection power is so
high it can kill even rigid spores)

2. for room of 10X10 feet you can use minimum of 500g of KMnO4 + 500ml of
Formaldehyde (when both are away they wont react, as soon as you mix it
fumes as volcano you should no be in the room and the surroundings, you
should run away from the room)

3. Seal the room to avoid the fumes to come out of the room

4. Wait for min 12 hours to act completely and efficiently

5. Wet the cloth with ammonia of 500ml and spread in the room for minimum of
6-7 hours to neutralize the fumes

6. Wipe the things with some disinfectant to clean and you can start your
work, I promise you won’t see any single mold for at least next 6 months and
year if you keep clean.



The only disadvantage is when fumigation is going on you can’t sit or work
for one day, maybe you can do this on Saturday night, So that it will be
ready for Monday. This is the usual method we do in CLASS-1 rooms and
CLASS-II, don’t look at the fumes or inhale and don’t near or it will harm
you lot take guidance from your professor and seniors while doing this. Any
thing else please feel free to contact.

--
Yours Sincerely,
*Naveen Vankadari*
Lab No: N209
Graduate Student,
Institute of Molecular & Cell Biology,
ACADEMIA SINICA,
128 Academia Road,section-2,
Nankang, Taipei-115
TAIWAN