Hi all
I used NP40 lysis buffer for HeLa cells and HEK293T cells and used the whole cell lysates to do Western blot. The main purpose was to determine what cell line express endogenous IGF-1R beta.
I used Bolt 4-12% Bis-Tris Plus Gels in Bolt® MES SDS running buffer and loaded 60 micrograms of the lysate (reduced and heated for 5 mins at 95 degrees). I ran the gel at 140 V for 60 minutes.
The primary antibody I used is polyclonal anti-rabbit IGF-1R beta (sc-713, 1/100 dilution).
The molecular weight of IGF-1R beta is 97 kDa however, the bands in HeLa cells appear a bit lower than expected. Therefore I am not very confident about whether the bands really represent IGF-1R beta.
Does anyone have the similar experience before? Can I argue that these bands seen in HeLa cells are IGF-1R beta?
Dear Yinan
- If bands run just under the expected molecular weight - as opposed to substantially less ( degradation of protein) - that could be due to differences in post translational modification compared to the reference line that the Mr was determined in
- Do you know what that ref line is from the antibody data sheet ? Furthermore could you make lysate with some of that line to verify an exact weight of 98kD ?
- If weight differs and you are really not sure then sometimes it is possible to purchase the synthetic peptide against which the antibody was made; precinct ate your antibody with this peptide; and then look for a reduction in signal (compared to antibody alone) which would confirm the band you are seeing is indeed specific
- On a more routine point did you include protease inhibitors in your lysis buffer when you made your lysate
- Finally, I will make the more general point that if you see bands of unexpected sizes and especially multiple bands trying an alternative source of primary antibody can be the difference between getting a Western blot to work; or not. This has happened to me many times
I think may be you can try gel with other percentage as well, if your protein is high molecular weight may be less percentage ...
I agree with the comments from Laurence and would like to add one more.
The ability of SDS to bind proteins might be affected by some non-ionic detergents such as Nonidet P-40. That might change a little bit the mobility of your protein in SDS-gel. For more information please see the link below.
https://cdn.shopify.com/s/files/1/0879/2408/files/GKE-5006.pdf?1350569799624999155
Thanks for replies everyone.
I did put a tablet of protease inhibitor in my lysis buffer.
I tried another Western blotting using the same sample from last time (which was frozen and thawed) but this time I loaded twice as much protein as the last time (120 ug) next to a lane in which I loaded 60 ug. Also, I used another anti-IGF-1R beta antibody (sc-9038).
As you see on the photo attached, both HeLa cells and HEK293T now show bands at 98kDa both with 60 and 120 ug of protein. The background is bit high maybe because I blocked with PBS-T instead of TBS-T.
Do you reckon the bands appear because of this new antibody I tried?
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