I have run my Crude extract protein from rice shoot sample and as per my observation I feel that my protein is getting diffused as lower part of my running gel. The gel solutions are freshly prepared as Tris pH 8.8 in resolving (10% Gel) while 6.8 as in stacking (5% stacking)

The buffer is composed with following component as final concentration of my protein extraction buffer

250mMSucrose

2mM Na2EDTA

10% glycerol

2mMDTT

1mM PMSF

5 mM B mercaptoethanol

0.25% Igepal(NP-40)

50mM Bis Tris Propane (pH 7.2-7.4)

Usually I used to crush my sample in liquid nitrogen followed by 15 mins cntrifuge at 12500 rpm approx.In last I collect supernatant of my solution which contains protein

The isolated crude extract is already quantified by Bradford and it is good in amount while this protein I have used various time for my plasma membrane associated H+ ATPase activity which has good result, even if this protein make it incubate for 90 degree for 5 to 10 minutes or leaving in room temperature for overnight it will make show 40-60% activity compare to non heated protein so I am not confused about the chances of degradation it was stored in -80 degree and it is checked recently for activity after 2 months of isolation.

My protein of interest is 45 kDa, 90 Kda and 215 Kda from my sample for western blot analysis but I am bit afraid that shall I go ahead with attached gel image with my sample as it seems me as diffused band. What I should do if it is really getting diffused after loading on my gel.Is there any possible contamination?

Starting 0.5 and 1.0% samaple are non boiled sample kept at 30 degree for 30minutes for sample preparation. Rest of the sample are heat denatured. The percent indicates the isolated protein from salt treated plant

Please look into my attached gel pic