Do you have a positive control for your 100 kDa protein? If not, that may be an important 1st step. There may simply not be enough of your 100kDa protein of interest in your sample to detect it, whether due to low expression or low AB sensitivity. Alternately, you might try a lower resolving concentration (I use 7.5% to detect ~100kDa mmNRF2), or reducing the methanol concentration in your transfer buffer a bit to improve transfer efficiency (these steps are probably unnecessary). You also have the options of loading more protein or using a higher concentration of 1' AB, though 60ug should be plenty if the protein is highly expressed and the AB is good.

It may be that there is an issue with the antibodies, particularly the primary, but I agree with Brian that there may not be enough protein available to bind to. The best way would be to use a positive control and see if that works, which means you could eliminate the Ab's being the issue. From there, you may want to try playing with the Ab concentrations, especially if the positive control is weak. 

Another quick test to see if the proteins are separating would be a Coomassie stain to make sure your protein bands are present in the gel to begin with. If that's not showing anything, you likely don't have enough protein to really show on the blot anyhow.

Try using prestained markers and making sure your high mw markers transfer fine.  For large proteins you need a little bit of SDS.  Try adding 0.01% SDS to your transfer buffer.  Monitoring your transfer with prestained markers is the best place to start to convince yourself that the protein really is on the blot.

Thanks   Bradbury. I will definitely try with 0.01 % SDS added transfer buffer. Hope it will work.

Dear Marx and Bower, Thanks for the suggestions. unfortunately I don't have any positive control for the 100 kD protein. I have tried with higher concentration of primary ab i.e 1:500 dilution and used the protein with concentration of nearly 60ug, so I don't think there is any problem with the concentration of antibody or sample.

all good advice from above seems like your blotting conditions are good  western blots can be tricky to optimise especially if not much is known about proteins and antibody is unknown it might be worth checking if your antibody at least detects and over expressed protein.

It is also Important to check the lysis conditions as your protein may be insoluble in your lysis conditions and therefore not much of it is present in your blot. this is something you may have to determine empirically 

is the protein from cultured cell lines? 

what is your lysis protocol ?

 Steve 

Yes Stephen  I have isolated the protein from a cultured neuroblastoma cell line using RIPA buffer with added protease inhibitor.

hello do you fractionate the sample by centrifugation after lysis with RIPA buffer then take the supernatant and pellet fractions and boil them in sample buffer ?I only ask as you make not be getting every thing soulable in RIPA buffer 

most of the time when collecting protein i wash cells with 1xPBS twice just to get rid of serum proteins in media then just add 1.5 dH2O and  3X sample buffer so for 6 well dish  250micoL water 250microL 3x sample buffer direct to the tissue culture plate heat this at 70 for 10mins then pass this 5-6 times through a 23g micro needle to make lysate less vicious.  

Sample buffer (0.3M DTT bromophenol blue 6% SDS 30% glycerol 0.18M Tris Ph6.8.)

Stephen,  I usually add RIPA buffer to the cell pellet after washing the pellet with 1XPBS, then centrifuge and collect the supernatant which contain the proteins.

while preparing the protein sample for loading to the gel, I usually boil taking the required amount of protein along with 5X loading dye ( composed of SDS, Bromophenol blue, Glycerol, Tris pH 6.8 , beta mercaptoethanol & MQ H2O) at 100 degree for 5min.

But  I think  the procedure you have mentoined have to be used  before isolation of protein. Kindly tell me the exact procedure you are telling about which might be helpful in my case.