During loading, sometimes some sample may end up in the neighbouring well or be washed into the buffer reservoir. That is one of the reasons why WB is, at best, a semi-quantitative method. You can reduce the effect by not overfilling the wells, by having enough glycerol in the sample and by careful loading.

I have good experience with Hamilton syringes with flat-tip needles. There are also special loading tips for Eppendorf pipettes, but I have never used them and cannot comment on their utility. It also helps to add a little phenol red to the stacking gel buffer (doi:10.1016/0003-2697(88)90297-7). The red dye makes the wells easier to see during loading.

you can use an internal (western-blot) standard (a house-keeping protein not to much expressed GAPDH? ) and then standardize the signal of your protein of interest relative to this internal control ....

Didier Poncet indeed I did, you can see it in the third photo, I apologise for not labelling it correctly. I used Actin as house-keeping protein

OK.... (I couldnot open your .qt files... ) and you are not satisfyed with the resukt? maybe the contact between the membrane and the gel was not good in some part of the gel so the transfert x-was uneven .... did you roll a clean pipette on the gel/membrane assembly to remove bubbles and have a good contact between gel and membrane...

yeah sure I did the normal procedure. I am sorry about the pictures, I shall upload them again

lane 4 and five for me it is a bubble problem; whether during transfert or during western blot