i doubt i have a problem in my extraction procedures .. i homogenize my brain in RIPA buffer NACl 150 mM , 25mM tris , SDS 0.1%, Sdium deoxycholate 1%, Triton x 1%, edta 5mM ... i use it in 1ul /mg of tissue. 2- i homogenize for 45 stokes in glass homogenizer and shake lysate for 2 hrs at 4 degree celsius in shaker and then i centrifuge and work on supernatent...i doubt i have sth wrong with my extraction? as although i have high conc of ptn no signals of beta actin in WB ...afterthis extraction i treat my sample in ratio of 1:1 in sample buffer ? is that too much amount of sds to add ? does high number of strokes could affect my ptn .plz help if ou have experience with ripa in tissues?
Hello Basma,
your protocol may have several potential problems:
1 Do you add a general protease inhibitor to your RIPA buffer?
2. Is your RIPA buffer pH'ed?
3. Can you decrease the number of strokes to homogenize the tissue?
4. Can you shorten the 2 hours to obtain the lysate? I work with cardiac muscle, but I need not more than 15 minutes to get a tissue lysate.
The next big area is your Western blot protocol. Many things can go wrong here (protein transfer, antibody, detection method and more). So my general question is, whether your western blot protocol ever worked? If not, you may need to troubleshoot your WB protocol.
Good luck
Basma,
First of all, if you are sure you are working with a good amount of protein, check the transfer efficiency before blocking the membrane and - is the transfer working fine - try a different antibody or optimize the incubation conditions.
If the transfer is not working, here are the suggestions:
1. How accurate is your total protein concentration estimate? What method did you use? Did you apply the dilution factor to the calculation? Did your normalizing control concentration units were the same as in the microplate reader program?
2. Check your calculations after diluting the samples with the loading buffer.
3. Did you follow the protocol for protein sample prep for electrophoresis such as adding bME to the loading buffer and heating the samples before loading onto the gel?
4. Did you see protein bands on the gel? including the area you expect to see b-actin? What protein markers are you using? Do you see the markers? Do you see the leading dye?
Also, here are a few comments in case you want to optimize the method:
1. Your ratio buffer/sample should be 10ul/mg or more, depends on the sample. The suspension should be liquid enough for optimal extraction and sampling.
2. As Gisela suggested above - shortening the time of the extraction may work well for the protocol.
Please keep us informed.
Good luck!
1-yes i add protease inhibitor complete tablete.
2- tris-hcl in ripa is ph 7.8, EDTA is of PH 8
3-i though large no of stokes in glass homogenizer is good for better extraction .am i wrong?
4- is it better to decrease no of shakeing also .i was told that this increase the desintegration of tissues to get better yield ...?
5- yes the protocol is working in lab ..forr other collegues testing membranal ptns. from cells and its working also we have made several changes and we became quiet sure it's about ptn extraction part ....
thank you so much doctors for your advices ...
1-i measured ptn conc by Qubit assay
it is 7mg/ml from one hemisphere when used PMSF only' when i used Comple protease inhibitor it is 12mg/ml
2- yes i heat my samples in 2xsample buffer for 60 degree celsius for 10 mins.
3- i always have my ladder transferred in semidry method either 1hr (RT)or 2h(with ice pack above) transfer.
4-i didnt stain the gel i always proceed to transfer and detection
Basma,
1-3 sound good. Try to stain the gel before and after the transfer with Coomassie and the membrane after the transfer with Ponceau S dye (Sigma P-7170). If all looks good, try a different a/b or optimize your visualization protocol if it's new.
Best!
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