Hello
I currently work on western blot, and I have some problem.
Would you please help me?
As you can see, my beta actin bands seems to diminish, not uneven.
Some membrane showed this kind of reducing, but some did not.
Most mysterious part is that two membranes which were incubated in one box showed different shape. One was even, and the other was not. I attached files of these membranes.
It didn't happen only two weeks ago, but now in this week, it does happen... It's really confusing.
I've considered the reason, but I cannot understand why it happened.
There are two reasons which I could concern.
1. Antibody distribution was uneven
- But, If it was a problem of antibody distribution, why membranes in the same box showed different trend?
2. Spreading ECL solution first on the left side, and then moving to right side was problem
- But, If it was a problem, why it didn't happen two weeks ago, and now bother me?
Here is a procedure
1. Cell lysis
- Lysis with RIPA+protease on ice bucket
- Scrapping cells with scrapper
- 30 minutes incubation in ice (to lysis more)
- Centrifuge: 16,000g, 40 min, 4 'C
- Take 90% of supernatant
2. BCA assay
- Check protein concentration of supernatant
- Make SDS-PAGE sample of 1 mg/mL (Dilution with 1X PBS, using commercial SDS-PAGE sample buffer)
3. SDS-PAGE
- loading 10 ug of protein (10 uL) on 10% SDS-PAGE gel (15 well)
- 100 V, 90 min
4. Transfer
- PVDF membrane, pre-activated with MeOH
- 100 V, 60 min with ice pack
5. Blocking
- 5% skim milk in 1X TBST solution
- 1 hr rocking, RT
6. Primary antibody
- 3% BSA in 1X TBST solution, antibody 1:1000 dilution
- O/N rocking, 4 'C
7. Secondary antibody
- 5% skim milk in 1X TBST solution
- 1 hr rocking, RT
8. ECL development
- Spreading ECL solution on membrane, and wait for 1 minute
- Remove ECL solution as much as possible by covering OHP film on the membrane
- Use increment mode of Las4000mini
I hope someone could save me...
Best regards
Hello Mingi Kim
I could possibly suggest the following:
1. The gel must have not been polymerized well. Ensure proper mixing of solution and give sufficient time for polymerization. Wash the wells with buffer prior to sample loading.
2. Decrease the loading concentration of protein on the gel. Loading 10ug may be a bit high.
3. Ensure that the PVDF membrane is uniformly wet.
4. Ensure that the volume of antibody used for incubation is sufficient for the membrane. The size of the box used for incubation should be slightly bigger than the membrane.
5. Use freshly prepared buffers and ensure that your chemical reagents are working well.
Hope this may help.
Best.
“Smiling”, where the central lanes run faster/farther than the end/edge lanes involves overloading of the lanes. Too high running voltage for your specific gel rig results in excess heat in a a pattern that affects the edges.
Dear Mingi, apart from above-mentioned explanations there are some more well-documented facts, like if the heat due to passage of current is not homogenous across the whole gel so may result in uneven bands and your loading buffer pH has a role in this case, because the resolving gel pH is usually around 8.8 and if the loading buffer pH raises to 8 and above so bands start to distort ultimately. Unbalanced salt concentrations in various protein samples may also lead to uneven bands and this is based on your each samples preparation, so to avoid or cross check this you can run your samples while keeping a lane free in between and these empty lanes wells should be filled alone with the buffer and then observe are there any uneven bands or not. Also, load always cool samples into the gel wells after the denaturation step. Remember that if you are making homemade gels then be sure that resolving gel surface should not be uneven at all and gel polymerization must be carried out properly and for this always look upon the status of TEMED and APS you are using, as these should be freshly prepared.
Regards....
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