I am having problem with detecting low molecular weight in western blot, desired kda size is 15 and 10 kda. i use 12% SDS gel, soak the PVDF membrane in MeOH for 30 min before transfer, transfer time 90V for 1:15 min, i just see a week bands on the membrane, what should i do?
Also consider using a PVDF membrane with a smaller pore size; our lab normally uses membranes with 0.45um pore size, but if we're specifically interested in low (10-15kDa) molecular weight proteins, we'll use membranes with 0.2um pore size. How many ug of protein are you loading?
send/ share your blot picture. How much protein and what antibody your are using?
10ul protein samples/well, i am using BDNF, IL6 and IL-1, the kda is 15, 23 and 26 respectively.
How you purify your samples? I will prefer only 5 min soaking of PVDF membrane in methanol. Although protein transfer look nice, try semi-dry transfer at 280mA constant current for one hour. Moreover, try nitrocellulose membrane. It is brighter than PVDF and give more nice bands. Also change your secondary antibody concentration. It looks much than require. You can try BSA in place of milk. BSA give more clear bands than milk. Wash membrane after 2ndry antibody at-least 3 times (5min) to effectively remove 2ndry. Please upload full photograph, it will give more idea what to do
Use 15% resolving and 5% stacking gels. Do not transfer for more than 90 minutes at 90V.
You can modify the primary and secondary antibody concentrations accordingly (use fewer secondary antibody concentration at 1:5000 perhaps, much like Waqar Ahmad has mentioned). You may also try loading in more volume of your protein sample (if you have enough) or increasing the exposure time during visualization, although the latter might yield more background noise.
Also consider using a PVDF membrane with a smaller pore size; our lab normally uses membranes with 0.45um pore size, but if we're specifically interested in low (10-15kDa) molecular weight proteins, we'll use membranes with 0.2um pore size. How many ug of protein are you loading?
Hi Afzal,
I will suggest you two things:
1- To follow Tris-Tricine (14-16%)-PAGE protocol for proteins
I would use 0.2 um PVDF membrane. Soak in methanol for 5 min not more. OR You can use regular nitrocellulose (0.2um) and no need to soak in methanol.
Wetting PVDF in meoh or etoh for 30s is plenty. Be sure to soak the wetted PVDF in transfer buffer for 5-15 min. However, it is even more important for transfers to thoroughly presoak the gel at least 15 but up to 30 min in transfer solution to remove all SDS. Using Dunn's transfer buffer with 10% meoh can improve transfer of proteins with high isoelectric points. Transfers will go faster in lower density gels and be better for spreading low weight protein bands as well (ie. 7.5% instead of 12%). The suggestions of 0.2 PVDF is also a good idea for small proteins. They can pass through the PVDF if transfer is too long or to strong. Nitrocellulose is also available in 0.2 micron size.
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