How to work out which volume is needed to coat the wells? My colleague said I needed to add 3.300 ml of water and aliquot it in tubes at 100ul each tube. Then to make a working solution add 900 ul of DPBS and add 50 ul to the wells. Is this correct?
When the product says you can aspirate the excess fibronectin is there a chance that you can remove all the fibronectin out leaving none in the wells?
I have all ready done this but I am worried that it is incorrect because the fibronectin doesn't seem to be working... although there might be other reasons why my ELISA is not working
also how do you keep the plates sterile in the different conditions
Your colleagues are correct, the exact volume is 3.125ml. Here is the explanation:
1. Area of a well in 96 well plate is 0.32 cm2, if you want to coat with 5ug/cm of fibronectin then you need 1.6ug of fibronectin per well in 96 well plate. (5/1*0.32=1.6ug)
2. 50ul volume is enough to cover a well in 96 well plate therefore you need 1.6ug/50ul for each well; which is equivalent to 32ug/ml concentration. (1.6/50*1000=32ug/ml)
3. If you are making stock solution at 10X then your stock solution concentration is 320ug/ml or 0.32mg/ml. (32X10=320ug or 0.32mg/mL)
4. If you have 1mg of fibronectin, you should dissolve it in 3.125ml of diluent recommended by Sigma to make a 10X stock solution. (1mg/0.32mg=3.125ml)
You have to incubate the plate with fibronectin for few hours at room temperature or overnight at 4 degrees. Yes, you can aspirate the excess fibronectin and let each well dry completely. You can rinse plate with diluent used for ELISA to make sure the unbound traces of fibronectin are removed.
As for as my knowledge is concerened, you have to coat x volume per well and not as x microgram/cm in a 96 well plate.
If you have 1 mg, dilute it in 1 ml and it becomes 1000 micrograms for ml. Later you can dilute it to 10 times or 100 timeswith water as per your requirement..............
Hi Melissa,
Use PBS pH 7.4, Carbonate buffer pH 9.5 for coating. You also may use Tris-HCl as coating buffer...coating buffer depends of protein you want to coat (try different conditions)...Note: avoid PBS in your buffers if plan to work with alkaline phosphatase-conjugated secondary antibody. In addition, avoid sodium azide if plan to work with peroxidase-conjugated secondary antibody. Try to coat different concentrations of your protein (serial dilutions: 1...10 µg/ml, for instance). Protein concentration depends of affinity of primary antibody, purity of protein, etc. Avoid frozen/thawed protein so prepare single-use aliquots of your protein. Coat 100µl diluted protein/well. You can try different incubation time/temperature conditions such as: room temp (RT) over nigh (o/n) ( with humidity), o/n 4C or 2h 37C...I prefer o/n incubation at 4C (better repetitivity between wells)
I hope it helps!
Your colleagues are correct, the exact volume is 3.125ml. Here is the explanation:
1. Area of a well in 96 well plate is 0.32 cm2, if you want to coat with 5ug/cm of fibronectin then you need 1.6ug of fibronectin per well in 96 well plate. (5/1*0.32=1.6ug)
2. 50ul volume is enough to cover a well in 96 well plate therefore you need 1.6ug/50ul for each well; which is equivalent to 32ug/ml concentration. (1.6/50*1000=32ug/ml)
3. If you are making stock solution at 10X then your stock solution concentration is 320ug/ml or 0.32mg/ml. (32X10=320ug or 0.32mg/mL)
4. If you have 1mg of fibronectin, you should dissolve it in 3.125ml of diluent recommended by Sigma to make a 10X stock solution. (1mg/0.32mg=3.125ml)
You have to incubate the plate with fibronectin for few hours at room temperature or overnight at 4 degrees. Yes, you can aspirate the excess fibronectin and let each well dry completely. You can rinse plate with diluent used for ELISA to make sure the unbound traces of fibronectin are removed.
can i dilute the fibronectin with DPBS without calcium or magnesium or should it be with these ions?
Hi Melissa, another tip for you - be gentle with the fibronectin! Don't vortex it or be rough with it as it can sheer the fibronectin into smaller fragments. I use that fibronectin at that concentration and it always works wells. I dissolve in water so that PBS should be fine. Good luck!
After coating a plate, you need keep it in 4 degree C for 6 - 8 hours. This is how we do for cytokines coating.
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