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Hi,
am a PhD scholar and working on HL-60 cells and arsenic. Now i wanna do mitochondrial membrane potential assay by flowcytometry using rhodamine 123 but unfortunately i can not find the protocol with rhodamine. please can someone help me by sending this protocol..... shall be thankful.
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New to MTT.... help calculate
05 September 2012 - 11:27 PM
Hi,
As I am new to MTT and a foreigner scholar in China, have to really strive the language barrier over here.... so am writting down my question here, I do know the protocol for MTT but could not find any help form internet that how to further calculate/interpret my readings for MTT I got on excel.
Can somebody please guide me that after getting the data (absorbance reading) on microsoft excel how can I actually calculate the cell viability rate. I mean what will be the formula applied or what i have to do....no nothing
....
Assay was performed in six replicates for blank, control and each concentration of drug.
I used a blank (no cell only mtt reagent+DMSO), control (cells+mtt reagent+DMSO), and various concentration of my drug in micromole (from 1um to 100um). Now I don't know what to do further with the absorbance obtained. I shall be thankful to the one who can teach me step by step calculations for mtt on excel to get the final results (i.e percentage survival).
As I am new to MTT and a foreigner scholar in China, have to really strive the language barrier over here.... so am writting down my question here, I do know the protocol for MTT but could not find any help form internet that how to further calculate/interpret my readings for MTT I got on excel.
Can somebody please guide me that after getting the data (absorbance reading) on microsoft excel how can I actually calculate the cell viability rate. I mean what will be the formula applied or what i have to do....no nothing
....Assay was performed in six replicates for blank, control and each concentration of drug.
I used a blank (no cell only mtt reagent+DMSO), control (cells+mtt reagent+DMSO), and various concentration of my drug in micromole (from 1um to 100um). Now I don't know what to do further with the absorbance obtained. I shall be thankful to the one who can teach me step by step calculations for mtt on excel to get the final results (i.e percentage survival).
No WB bands for H2AX at all...!
13 August 2012 - 10:35 PM
Hi,
I am a PhD scholar working with HL-60 cells and I am trying to induce DNA damage to them and detect gamma H2AX proteins (15kDa) by western blotting using a well-documented toxin, Arsenic.
However, instead of seeing a band at the expected 15kDA region, I've detected nothing after repeating the experiment several times. However, my actin band appeared whenever I detected for H2AX and actin simultaneously but surprisingly, there were no band at all for H2AX...! Only once i got some light and merged bands at 15kDa but at that time no actin band appeared..!
I am very sure that the H2AX proteins are in the cells because I am using high molar concentration of arsenic that is sufficient to induce DNA damage.
where i am going wrong:
Running the gel at wrong voltage (100V) and time (2.5hrs)?
Using wrong PVDF membrane size (0.2uM )?
Transferring proteins at wrong current (200) or for wrong time (1h and 15min)
Using wrong dilution of primary antibody (1:500)?
Using wrong dilution of secondary antibody (1:10000)?
Here is my protocol:
1. Lyse the cells. centrifuge at top speed for 30mins at 4deg Celsius to remove cell debris.
2. Quantitate amount of proteins and load 20ul of protein sample onto a 13% gel. Run gel at 100V for 2.5hrs.
3. Transfer proteins to a 0.2uM PVDF membrane at1h and 15min.
4. Block membrane in freshly prepared 5% milk (in TTBS) at room temp for 1 hr.
5. Incubate membrane with anti-phospho-histone H2AX, diluted 1:500, overnight at 4deg Celsius.
6. Wash with TTBS (3X). Incubate with Rabbit HRP conjugated IgG (diluted 1:10000) for 1hr at room temp.
7. Wash with TTBS (3X) and detect with Immobilon Western Chemiluminescent HRP Substrate.
Please help me out with this problem of mine..
I shall be very thankful to you.
I am a PhD scholar working with HL-60 cells and I am trying to induce DNA damage to them and detect gamma H2AX proteins (15kDa) by western blotting using a well-documented toxin, Arsenic.
However, instead of seeing a band at the expected 15kDA region, I've detected nothing after repeating the experiment several times. However, my actin band appeared whenever I detected for H2AX and actin simultaneously but surprisingly, there were no band at all for H2AX...! Only once i got some light and merged bands at 15kDa but at that time no actin band appeared..!
I am very sure that the H2AX proteins are in the cells because I am using high molar concentration of arsenic that is sufficient to induce DNA damage.
where i am going wrong:
Running the gel at wrong voltage (100V) and time (2.5hrs)?
Using wrong PVDF membrane size (0.2uM )?
Transferring proteins at wrong current (200) or for wrong time (1h and 15min)
Using wrong dilution of primary antibody (1:500)?
Using wrong dilution of secondary antibody (1:10000)?
Here is my protocol:
1. Lyse the cells. centrifuge at top speed for 30mins at 4deg Celsius to remove cell debris.
2. Quantitate amount of proteins and load 20ul of protein sample onto a 13% gel. Run gel at 100V for 2.5hrs.
3. Transfer proteins to a 0.2uM PVDF membrane at1h and 15min.
4. Block membrane in freshly prepared 5% milk (in TTBS) at room temp for 1 hr.
5. Incubate membrane with anti-phospho-histone H2AX, diluted 1:500, overnight at 4deg Celsius.
6. Wash with TTBS (3X). Incubate with Rabbit HRP conjugated IgG (diluted 1:10000) for 1hr at room temp.
7. Wash with TTBS (3X) and detect with Immobilon Western Chemiluminescent HRP Substrate.
Please help me out with this problem of mine..
I shall be very thankful to you.
