The three images attached here are for 3 different proteins probed at the same time. The first one is GAPDH. Does anyone have any idea why (1) Despite reasonable Bradford assay results, GAPDH seems to vary significantly across samples? (2) The individual bands seems to give a smiling shape, even though I am confident that it is not due to gel overheating? (3) The bands tend to bend upwards, even though they are of the same molecular weights (refer 3rd image)?
Here is a brief summary of my procedures:
(1) The proteins were extracted using RIPA buffer.
(2) The lysate were quantified using Bradford assay.
(3) For each sample, 40ug/15uL proteins were prepared (Because there are three proteins to be quantified, a 120ug/45uL sample was prepared).
(4) An equal volume (45uL) of 2x Laemmli sample buffer was added to each sample and boiled at 95*C for 10 minutes, then placed on ice.
(5) The SDS-PAGE apparatus were set up, and 25uL of each sample was added to each well in a 10% gel (the samples were briefly resuspended by pipetting before being loaded).
(6) The tank was surrounded with ice, and the electrophoresis ran at 80V for the first 10 minutes, followed by 120V for approximately 2 hours.
(7) After the SDS-PAGE was done, the proteins were transferred to a PVDF membrane using wet transfer at 100V for 1 hour and 30 minutes. The tank was surrounded with ice, and an ice pack was placed inside the tank.
(8) After the electrotransfer is done, the membrane was blocked with a blocking solution for 1 hour.
(9) After blocking, the membrane was washed with TBST three times (I now know that this washing step probably wasn't a very good idea), before staining with primary antibody at 4*C overnight.
(10) Following primary antibody incubation, the membrane was washed three times with TBST before staining with secondary antibody for 1 hour at room temperature.
(11) Finally, the membrane was washed three times with TBST and the blots were viewed.
I'm pretty much at a loss here, so any suggestions would be greatly appreciated.
The defects in sample stacking which is evident in your picture2 & 3 can be due to ionic strength of buffer used for runing of gel as well transfer to membrane. suggest you to make fresh buffer and doubly check pH.
I would suggest make protein sample separately for all the three protein instead of pooling to avoid variation among samples.
yeah.. you are right, please don't wash after blocking, simply add primary antibody directly in to blocking buffer.
All the best for your future experiments
RIPA buffer contains a lot of detergent, which interferes with the Bradford assay. This may be why you see different amounts of protein bands in the different samples.
Besides what is mentioned above, I would use 6x Laemmli buffer and freshly made running and transfer buffers. Also check methanol grade if using it for transfer. I would consider a test using nitrocellulose membrane and Ponceau S staining to check if your system is fine up there.
Hi Jeremy
I suggest you go ahead in parts:
The first question that arises is: did you measure total protein in triplicate?
Then work only on the gel until you get a good image of the bands, after staining with comassie. In this image you can quantify total protein by densitometry each column of the gel (all the bands of the column) to check if there is large variation between samples. After having this overcome if I see rational continue with membrane transference. After having the transfer ready, through confirmation with ponceau red staining, you can follow the use of antibodies but standardizing only one protein.
Regards
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