I did western blotting for a 55kDa and 97kDa protein and used 1:5000 dilution of Primary antibodies and 1:10000 dilution of secondary antibodies. The blot was developed using DAB and i got a good band for 55kDa protein whereas the 97kDa protein detection was very faint.
Since the protein is nearly 100kDa, is it necessary to add 0.1% SDS in the transfer buffer. The transfer was done at room temperature for 2 hours at 90V (wet transfer).
If you are not expecting differences in the expression of the two bands, acting on the transfer protocol may give some improvements:
- First of all I strongly suggest to operate at 4°C instead than at RT for the high amount of current that is generated in the wet transfer procedure at 90V. You may also consider to lower the voltage to 50 and increasing the transfer tome to overnigth always at 4°C.
- 0,1% SDS may be added, so as methanol may be lowered to 10% as this will avoid in-gel precipitation of large proteins.
- A low concentration gel (less than 8%, we have good results with 7,5%) will improve larger proteins transfer across the gel to the membrane.
These are some simple steps which may help.
How is the transfer performance of the markers you run together with the sample?
If you are not expecting differences in the expression of the two bands, acting on the transfer protocol may give some improvements:
- First of all I strongly suggest to operate at 4°C instead than at RT for the high amount of current that is generated in the wet transfer procedure at 90V. You may also consider to lower the voltage to 50 and increasing the transfer tome to overnigth always at 4°C.
- 0,1% SDS may be added, so as methanol may be lowered to 10% as this will avoid in-gel precipitation of large proteins.
- A low concentration gel (less than 8%, we have good results with 7,5%) will improve larger proteins transfer across the gel to the membrane.
These are some simple steps which may help.
How is the transfer performance of the markers you run together with the sample?
I am not expecting any expression differences since these are purified fractions and a known amount was loaded. The marker was fine and clearly visible.
Hi Eniyan
For transferring "big" proteins (bigger than 100 KDa) we remove completely the methanol from the transfer buffer, and we obtained good results.
good luck
Iñaki
I concur with Iñaki,
actually methanol is only necessary if using nitrocellulose.
If using PVDF, methanol can be removed from the transfer buffer.
Hi Eniyan,
First you must make sure about protein blotting, so after protein blotting procedure, its better stain nitrocellulose paper with Ponceau S that may be used to prepare a stain for rapid reversible detection of protein bands on nitrocellulose or polyvinylidene fluoride (PVDF) membranes (Western blotting), as well as on cellulose acetate membranes. If you have any problem with protein blotting, this method will show you.
Good Luck
Jafar
when trying to blot my 150 kDa and actin 42 kDa by tris/gly/15% methanol, 100 V for 4 hours the smaller one blotted great in nitrocellulose whereas only small amounts of my large protein blotted. But when i added 0.01% SDS to the transfer buffer 100V/3 hours, great large protein blotting whereas mostly all my actin blow over as i stained the gel after blotting and no proteins were detected in all lanes. what do you suggest to blot both. kindly help me.
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