i working on mice. i have to extract retina for doing western blot. unfortunately i am not getting any bands. my question is that how much we should use lysis buffer and how long time we need to keep it in 4 C
Eye are robust to isolate and you require stronger lysis buffer. I would recommend using RIPA buffer and sonicate ur sample sonicating for 20 s, and then centrifuging for 15 min at 14000 rpm at 4 °C.
check this study as well
"An easy, rapid method to isolate RPE cell protein from the mouse eye".
Hi, Maryam.
To add to the previous answer, I would try a proportion of ~5mg of tissue per ~300 uL of lysis buffer (if I am not sure, I weight the samples previously). I work with this proportion to avoid overdilution of samples. The minimum recommended protein concentration for WB is 0.1 mg/mL, and good concentrations are within the range of 1–5 mg/mL.
If you are asking for how long lysis buffer can endure storage and keep its properties, I am not sure. If your experiments are too far apart from each other, you will be safe making a new extraction buffer before each one. During the extraction, I would recommend to kept it at 4 °C until the procedure is done.
Good luck!
actually mice retina is very small. so some time its difficult to measure the weight.so now i am trying to reduce lysis buffer from 30-40 ul.
We optimized after several trial the optimal amount: 75-100 ul per retina.
The secret is to keep in lysis buffer (on ice) for at least 40 min vortexing for few seconds every 5 min.
I spin the sample after the processing, and take supernatant.
With this procedure you can obtain a concentration between 2.0-3.0 ug/ul, and a final volume of at least 50 ul of lysate, good therefore for a couple of lanes.
If you can , I would suggest to use two retina per sample (than you can around 4-5 lines).
Please note: this protocol is per DISSECTED RETINA that means: no lens, no sclera, no cornea, JUST retina.
I hope it helps
Thank you very much, I have sorted out, the problem wasn't lysis buffer. it was second anti body problem.
I bet! I realized later your post was not exactly recent lol!
I wrote down in case you or other scientist will need to do a similar procedure.
Thank you for your reply!
Lanfranco Leo Hi,
May I know the composition for your lysis buffer please?
Thanks
Maryam Chudhary
Hi Maryam, Can you please share your lysis buffer recipe and protocol you followed for retinal tissue lysis.
Thanks
Sowmya Bekshe Lokappa Sowmya Bekshe Lokappa I use the commercially available RIPA BUFFER from cell signaling (cat # 9806) + a protease inhibitor cocktail (freshly made) at 1X concentration (cat# 11697498001)
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