Hi,
I am trying to perform IP with HSF1 antibody from liver tissue but no success so far. I prepared my tissue lysate in RIPA buffer and I am using 1mg protein for IP and bead used is protein A/G agarose beads from CST. I have done IP with HSF1 antibody from diffrent companies as shown in attached image and Immunoblotting is done with HSF1 antibody from Enzo life sciences and I am always getting a High molecular weight band (around 250-300KDa) along with light and heavy chain from IgG but no band at 75-80KDa (mol wt of HSF1 Ab). Please help me regarding this.
Thank you
Hi,
I'm not an expert in the field, but I can give you an opinion from a biochemical viewpoint. My first impression would be that your Enzo Ab is not working at all, the signals in the input lane (at least 3, all same intensity, very weak) are probably just background, what you indicate as HSF1 is too small, the band is typically at the height of the 80 kDa marker band, not 60. I believe a good Ab for western blot would be the CST 4365 rabbit polyclonal (it's actually pretty good for everything, as often the CST antibodies) [see Qiao et al, JCB 2017, for example]...
Troubleshooting the high mol weight bands might be more difficult, they appear only in the IPs, so it doesn't seem to be related to the lysis (although I don't know how much input you loaded, maybe very little...?), but more to the processing during or at the end of the IP. In general these effects on denaturing gels are due to some protein aggregation, but they normally look less defined, less band-like.
Dear R. Trivedi,
Try to switch to a monoclonal antibody for the WB detection with preferences to those originated from a different species than the IP antibody and binding to a different epitope than the IP antibody.
Good luck!
hi
it seems your IP not work well. you can try to this step with carefully for reducing background and inhibit to capture tissue IgG by A/G beads :
1- wash 1mg liver tissue 3 times with 5 ml PBS buffer ( reduce blood and immunoglbulins)
2- do it( Cell Lysate Pre-Clearing step ) with carefully >>
capture tissue IgG by A/G beads before run the original IP .
Pre-clearing the lysates :
Pre-clearing the lysate can help reduce non-specific binding and reduce background. However, if the final detection of the protein is by western blotting, pre-clearing may not be necessary unless a contaminating protein is interfering with visualization of the protein of interest.
It is important to make sure that as much of the normal serum is removed as possible, as this will compete with the antibody against the antigen of interest. To check for this, a test can be done with lysis buffer instead of sample, performing all pre-clearing steps as above.
Running a gel of the resulting supernatant and staining with Coomassie will reveal if the serum Ig is being removed effectively. If serum has not been sufficiently removed, bands will be present at 50 and 25 kDa for heavy and light chains; its presence may contribute to a weak immunoprecipitation. Consider either decreasing the amount of serum or increasing the amount of beads incubated with your samples in the pre-clearing step.
Washing
Wash buffers
- 10mM Tris; adjust to pH 7.4
- 1mM EDTA
- 1mM EGTA; pH 8.0
- 150mM NaCl
- 1% Triton X-100
- 0.2mM sodium orthovanadate
- Protease inhibitor cocktail
Store up to 6 months at 4°C. Immediately before use add protease inhibitor.
good luck
@ Reza Hadavi
Hi
1) I started with 30mg tissue and washed 3-times with PBS+PIC. I also preleased my cell lysate with beads for 1hr at 4 degree C.
Sorry, it seems I did not understand your few comments correctly.
Add either 50 μL of off-target antibody of the same species and isotype as the immunoprecipitation antibody, or normal serum (rabbit is often preferred, see Harlow and Lane, page 243) to 1 mL of lysate. Incubate for 1 h on ice
2) In the above comment, are you trying to say that I should clear the lysate with off target antibody and normal IgG along with beads simultaneously to get rid of IgG and non-specific binding of beads from lysate?
I also did not get the following statement
To increase the yield, the beads can be washed 1 or 2 more times in lysis buffer, and the supernatants collected together
3) Could you please explain it again?
It is important to make sure that as much of the normal serum is removed as possible, as this will compete with the antibody against the antigen of interest. To check for this, a test can be done with lysis buffer instead of sample, performing all pre-clearing steps as above.
I did not get the above comment as well especially lysis buffer instead of sample and how it would help?
4) I am using the same buffer for washing steps.
Thank you so much.
Rachana
@ Irina Donahue
OK, I will follow your suggestion. Thank you so much.
Rachana
@Pietro Pilo Boyl
Hi,
I loaded 10% input. I will also try this HSF1 antibody from CST.
Thanks for your suggestion.
Rachana
hi
In my opinion, your antibody does not succeed in HSF1 capturing, due to the high levels of tissue immunoglobulin in the liver, you should before the run "IP" remove (liver IgG) from the tissue lysates by protein A/G agarose bead .Because high levels of tissue immunoglobulin can compete with anti HSF1 antibody to binding the protein A/G agarose bead so there is no other empty capacity in A/G bead to absorb the anti HSF1 antobody.In the previous comment ,I provided Abcam company protocol for you . But simply before you start orginal IP with HSF1antibody you should be removed liver IgG from the tissue lysate by a certain amount of the A/G beads from your lysates(clean up your liver lysate from tissue IgG).
good luck
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