Dear All,
I am trying to express a large protein (220 kDa) with a GST tag. I performed Western Blot to check for the protein of interest. Now after using monoclonal antibody (Mouse Ab), I have used Mouse secondary Ab and check into the IR imager (as the secondary Ab lit up in IR). I also used Positive control of the protein that doesn't show any signal this. However almost all the bands have shown signals into the imager. I am attaching the results with indication of what is what. From left, 1 lane is the marker, second one is the whole cell lysate, 3rd lane is the supernatant, 4th and 5th are two different positive controls. I have stringently followed the standard steps for the western blott. All your suggestions are welcomed to improve my result. Thank you in advance.
Dr Prakash,
Hi,
Can you label the markers at their respective sizes. Expressing such big recombinant protein isn't an easy task. Even I am working on similar size protein. I have prepared in six fragments instead with a GST tag? How sure are you that the construct you prepared is working. Also use anti-GST antibody to reconfirm your result (including a positive construct).
May I know the preparation protocol in brief?
Although I have prepared full length with HA-Protein-HA tag. But its expression is very low (maybe a nature of it).
There seems to be a problem with your antibodies (either primary or secondary), as you stain total protein. This could be for example insufficient blocking and/or too high an antibody concentration. Perhaps you should ask somebody with experience in western blotting to watch your technique, just in case you make some trivial mistake.
Once that has been sorted out, I'd try to assay for production of the protein (in whole cells put directly into sample buffer), the lysate, the pellet (inclusion bodies!), the flow through of the affinity column (may not bind) and the eluate. I'd try an electrophoresis method for large proteins (doi:10.1021/bi00789a030, doi:10.1002/elps.200900657) and perhaps an anti-GST primary antibody in addition to the one against the protein of interest.
Good luck!
With a such big protein you are testing, I have the following concerns:
1. What percentage PAGE gel you are using? For a protein of 220 KD, gradient gel is suggested, such as 5%-20% gel.
2. For large protein, transfer from gel to the membrane is very insufficient . Extending your transfer time is recommended.
3. Check your membrane binding capacity. PVDF is recommended.
4. Based on your image, too much protein was loaded.
5. Do dot blot of your positive controls to test whether your detection antibodies work.
Good luck!
Hi Prem ,
I have already optimised my 220 kDa protein from mammalian cells. I use 8% and 6% SDS PAGE and transfer it for 3hrs or overnight. The protein is efficiently transferred and blot are perfect. But the only thing I doubt is that Is his construct actually expressing. This is a problem I have already faced and retried until I got its expression which is quite low and can't be used for pull-down studies. Thanks
Pratibha Srivastava The upper most band is of 250 kDa.Yes, I wanted to be sure whether the construct works well and this was my first test. I want to purify the the full length as my objective is to look into the whole structure using cryoEM. It would be great if I succeed in expressing in E.coli in atleast decent yield.
Prem Prakash Perhaps for me I think your construct isn't expressing well. Also, it depends of how much protein you have loaded.
Try one more time transfecting 20ug of construct and you can load 300ug of the protein. Blot it using anti-protein and anti-GST to avoid confusion. Make sure you have positive controls for each. Good luck
Dear Pratibha Srivastava,
Thanks for your reply. But I'm a little bit confused how your work is related to Prem Prakash's experiment, since I was trying to help Prem Prakash troubleshooting his Western-Blot result. According to his Western-blot image and description, his positive control didn't show any signal and its size is 220KD, the first two questions that should be asked are: 1) is the transfer succeed? 2) does the detection antibody work? Therefore, I gave some suggestions to look into his assay.
Of course, if his positive control showed signal, meaning there is no problem with his Western-blot assay, then the transfection efficiency and expression levels of the targeting protein should be considered.
Hi Prem . We can’t ignore the fact that overexpressing recombinant protein in E.Coli create unbalance proteostasis, as a result many endogenous proteins rapidly increase in cells. for example chaperones of high molecular weight. In such case, secondary antibodies can be easily adsorbed by such proteins if antibody titration is too high or absence of condition which reduces non-specific interaction ( for eg. nonreactive protein in blocking buffer, Triton-X detergent in washing buffer). Secondly, their is no signal from postive control, getting good IB results by using monoclonal ab is quite challenging because detection is based on recognising single epitope and sometime such epitope
get masked by the fusion of tag or other proteins . I hope it’s not be your case . you can try below steps as I usually follow for monoclonal ab.
1. Load protein in range of 150-300ug. if IB detection is poor
2. Blocking 1%Milk in 1xPBS 2-3hrs at 4 degree
3. Dilute primary ab in low dilution for eg. 1:200 in 1% Milk, overnight incubation at 4 degree
4. Dilute secondary ab in higher dilution 1:2000 in 2-3% Milk, Incubation at RT for 1 hr. Then Washing in PBST buffer 4 times 5 min each ( this steps can be optimized depends on signal/noise ratio)and then Immunoblotting
I hope this can help you. All the best !
Arpit
I have looked at your image. Only first 3-lanes are visible. My first impression was that this is the image of SDS-PAGE and not western blot. There could be several problems: a) with primary antibody; b) improper blocking; c) not having a good positive control. The only suggestion that I can give is to obtain a working primary antibody from other investigators/collaborators who have worked/published their results with your particular protein and then compare that with your antibody. For your size of protein your should run 6% or 7% gel. Good-luck.
I agree, most propably your antibodies work not optimally or is not specific which is awell known problem found in various publication (e.g. see Baker, M. NATURE. VOL 521 (21 MAY 2015)).
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