Antisera from the Lindberg Laboratory

Antisera2016

Lindberg Lab Antisera Descriptions (2016, IL)

The first number (bolded) is the rabbit number; on each tube, it will be followed by a B for Bleed, then the second number, which is variable since it is the bleed number. F denotes the Final bleed. All numbered antisera (except numbers 45 and 46 and s43 and s44) are raised to peptides 10-15 residues in length, usually c-terminal, conjugated to keyhole limpet hemocyanin (Pierce Chem) with ECDI (with some exceptions).

Please look up the full reference (in the CV listed on this same website) and CITE IT when using one of these antisera. Thanks!

Note after Katrina- samples of all antisera were rescued at three weeks in our famous guerrilla raid. While many were moldy, some had azide and were fine (you can guess what the moral is here). We have partially purified IgGs from about half of these antisera using ammonium sulfate precipitation. We did this to decrease the number of small bottles from different bleeds, which were taking up too much space. Both previously moldy and non-moldy antisera appear to work well for blotting; other uses have not been tested thus far, but they will probably be fine. We also have certain, but not most, preimmune sera which suffered identical fates.

1 & 2: Mouse PC1, mature N-terminus. 2 is most commonly used (and now is out)- Good for immunoppt and Western blotting; ICC; RIA titer over a million. Reference Vindrola and Lindberg Mol. Endo. 1992 and Hornby et al Neuroendocrinol. 1993 (for ICC). Affinity Bioreagents carries a similar antiserum, tested by our lab. The peptide immunogen has been supplied to the Mains and Devi labs and they have raised similar antisera. Affinity Bioreagents carries a similar antiserum raised using our antigen and tested by our lab. We have a lot of antiserum 1; 2 is out. The peptide sequence is svqkdsaldlfndpmwn, conjugated to succinylated hemocyanin.

3: Mouse PC1/3 C-terminus- this one does not see the 66 kDa form. Vindrola and Lindberg Mol. Endo. 1992 and Hornby et al Neuroendocrinol. 1993 (for ICC).The peptide used was: 714-726 (lqalmdilneen), conjugated to succinylated hemocyanin.

4 & 5: Mouse PC2 C-terminus. 4 was most commonly used. Supplanted by 18 because we ran out. Good for immunoppt, Western blotting and ICC. Reference Shen et al, 1993. Note that Affinity Bioreagents carries a similar antiserum raised tested by our lab.This is raised to the last 12 residues of mouse PC2 (YERSLQSILRKN) coupled to succinylated KLH.

6 & 7: Mouse PC2 mature N-terminus, gyrdineidinmnd. 7 is most commonly used. Reference Blanco 2014 paper.

12 & 13: same 7B2 epitope originally used by the Seidah group (internal, residues 23-39). 13 most commonly used. All vertebrate species. Blots recombinant stuff, but not as well as the Martens monoclonal. Good for RIA, Immunoppt. Reference Zhu and Lindberg 1994. Note- it is VERY hard to see 7B2 in tissues and even on blots unless you have an enriched tissue like pituitary or adrenal. Use a radioimmunoassay!

14 & 15: Human C-terminus (residues 18-31 of CT peptide) of human 7B2. HUMAN- SPECIFIC. Not published.

16 & 17: Human CT peptide 1-16. Requires CPB removal of basic residues to see immunoreactivity! Prefers human, but works with mouse. Good for RIA. Reference Zhu et al 1996 PNAS.

18, 19: New mouse PC2 antiserum. 18 most commonly used. See antisera 4 and 5 for characteristics. Same epitope and preparation.Reference Muller et al JCB 1997.

23 & 24: C-terminus of rat/mouse 7B2 (18-31). 23 is most commonly used. Good for RIA. Reference 1996 Zhu PNAS.

26 & 27: mouse PC2 propeptide. 26 most commonly used. Antigen is His-58 to Asp 80 of mouse proPC2 coupled to hemocyanin. Good for RIA and immunoppt. Reference Muller et al 2000 JBC.

35 & 36: Drosophila PC2 antiserum (C-terminus of Drosophila sequence). Reference paper.Works for ICC, immunoppt, Western blotting. Hwang et al, 2000 JBC. This is a good neuroendocrine marker in Drosophila. Ask us for it!

37 & 38: QERARAEAQEAED antiserum to mouse proSAAS. 38 most commonly used. Blots poorly and does not immunoppt; not good for ICC. Reference Sayah 2000 J. Neurochem. paper.

39 & 40: LENPSPQAPA antiserum to mouse proSAAS CT peptide. 40 most commonly used. Works for immunoppt but not great for blotting of endogenous stuff (probably due to removal of CT peptide?). Not good for ICC. Reference Sayah 2000 J. Neurochem paper

41 & 42: raised to ACTH 1-18. Used for immunoppt to replace antiserum JH93 of Dick Mains. 41 is used most. Recognizes POMC, ACTH and cleaved ACTH (alpha MSH). Reference Fortenberry et al, JBC, 2002

43 & 44: N-terminus of mouse SAAS (ie, the SAAS peptide itself; SLSAASPLVETSTPLRL coupled to KLH). Good for ICC. See Jarvela et al PNAS paper, 2016.

45 & 46: Raised to recombinant His-tagged 21 kDa mouse proSAAS. Good for Western, IP and ICC. First published by John Hutton, for pancreatic islet ICC (see his reference)(image also present in islet CJ Rhodes paper). See also Hoshino et al 2014 J. Nueroschem paper; and 2016 PNAS paper (Jarvel et al) for brain/neuron staining.

47, 48: TMEM66, also known as SARAF (and “Mike’s protein”)..never used…CQNKGWDGYDVQ is the peptide, KLH the protein. Ask us for it.

49, 50: raised against C. elegans 7B2- never used- ask us for it! ESLQKILEENNMHANT is the peptide, KLH the protein

91,92: Polyarginine antisera (raised to D9R coupled to KLH). Not published yet. Works well in elisa (for example in determining blood levels of D9R)

93: Proghrelin C-terminus (EEAKEAPADK) Not published. Great for Western blotting.

94: Ghrelin peptide c-terminus(VQQRKESKKPPAKLQP) Not published. Great for both ELISA and RIA.

s43, s44: Seth’s human furin polyclonal (raised in rabbits at LSUHSC against our recombinant human furin). Excellent titer in elisa- 100K for #s44!); not published. We also have some human furin monoclonal from this lab. No protein carrier. Not very much left.

95,96: Octanoylated ghrelin- a small peptide containing the acyl group. raised to GSS(octanoyl)FLSPEHQ; sequence is conserved among vertebrates so both human and mouse. Some cross-reaction with unacylated ghrelin. Works in ELISA against the acylated peptide. Covance 2007.

5519, 5520: Clec3a- Raised to sequence CSFLNWDRAQPSG-amide from mouse/human CLEC3A. Conjugated to hemocyanin via Cys; never used. NEP made, 2008?

6426, 6427: Augurin- sequence EGPVPSKTNVAVCG Aug-1 peptide, Augurin 42-53 conjugated to hemocyanin via cysteine. Doesn’t recognize cell products by blotting or immunoprecipitation-?! NEP made, 2008? Not published

MD235, 236: Augurin, raised against His-proaugurin (recombinant human), works in Western blotting only against human recombinant proaugurin and products, not cell-synthesized material. Covance; 2009. See Ozawa augurin paper.

MD243, 244: C-terminal peptide of human Augurin2 (137-148), FRHGASVNYDDY, conjugated to succinylated hemocyanin, works in ELISA only against immunogen (ie the above peptide). Covance, 2010 See Ozawa augurin paper.

MD262* and 263: Human C4orf26 conjugated to hemocyanin; peptide is QEEVFTLPGDSQ; works in Western blotting against proC4orf26. Covance; final bleed 8/22/11. ELISA against peptide immunogen alone does not work. # 262 most useful for protein recognition via Western blotting, but peptide products are not visible. Not yet published.

MD275 and 276*: mouse FGF23 C-terminal antiserum, CSRELPSAEEGGPAASD. Raised to the same antigen as previously published C-terminal antiserum- used cysteine coupling with maleiimide. Covance made this in the Fall 2012. 276 is best; we have a production bleed. Confirmed working by Western blot using recombinant material; see 12-14 kDa band. (Note that we now know that these are likely phosphorylated segments= the labeled phosphate resides in this segment!) Immunoreactivity might be improved if treated with alkaline phosphatase.

MD279, 280– human C-terminal FGF23 antiserum, CSQELPSAEDNSPMASDP. Winter 2012. Again, we now know this epitope contains phosphorylated residues! We have not been able to show this antiserum works yet… Need to treat samples with alkaline phosphatase prior to blotting?

MD283 and MD284– to phosphorylated human C-terminal FGF23. June 2013. Made to CSQELP{pSER}AE (Genscript) conjugated to KLH with ECDI (Covance). This antiserum reacts well with recombinant human FGF23, and also with a Flag blockade mutant transfected simultaneously with FAM20C plasmid (but not with dead kinase-encoding plasmid). MD283 mostly used. Protein A-purified AB is 0.20 mg/ml and the Affigel Blue/NH4SO4 material is 0.48 mg/ml. See JBMR paper Lindberg et al.

 

STEINER ANTISERA: (bequest from Dr. Donald Steiner)

Human PC5: epitope is DYDLSHAQSTYFNDPKWPS (never published?) probably used in ghrelin paper (see below) to confirm PC5 ko.

Ghrelin antiserum (mouse; see Steiner ghrelin paper PMID:17050541). Good for tissue staining.

Obestatin antiserum (epitope unknown)- see Steiner paper.

OLDER ENKEPHALIN ANTISERA:

Lucy (or 215): raised to Leu-enkephalin coupled to thyroglobulin with glutaraldehyde. Good for RIA and Westerns. Recognizes Leu-enk and amino terminally extended forms only. Reference: Lindberg, I., and Dahl, J.L. (1981) Characterization of enkephalin release from rat striatum. J. Neurochem. 36, 506-512.

NZA- raised against Met-enk-Thyroglobulin AND met-enk-BSA

NZB- raised against Leu-enk-thyroglobulin AND leu-enk-BSA

Pat (also known as 207) raised against Leu-enk-thyroglobulin

Cass and Dick: raised to Met-enkephalin-RGL coupled to hemocyanin with ECDI. Good for immunoprecipitation, RIA, and Westerns, Cass used most often. Recognizes the octapeptide and amino terminally extended forms. Reference: Lindberg, I., and White, L. (1986) Reptilian enkephalins: implications for the evolution of proenkephalin. Arch. Biochem. Biophys. 245, 1-7.

Betty: raised to Met-enk coupled to hemocyanin with ECDI. A low titer but reasonably sensitive Met-enk antiserum for RIA. Not published.

Xandra: raised against Met-enk-RF coupled to succinylated hemocyanin with ECDI; recognizes proenkephalin, Peptide B and Met-enk-RF. Good for immunoprecip, RIA, and blotting. Reference: Lindberg, I., and Thomas, G. (1990) Cleavage of proenkephalin by a chromaffin granule processing enzyme. Endocrinology 126, 480-487.

Yolanda: raised against bovine proenkephalin Peptide B (C-terminal fragment of proenkephalin) coupled to hemocyanin with ECDI ; recognizes Peptide B, but directed against the non-Met-enk-RF portion of the molecule. Reference: Lindberg, I., and White, L. (1986) Distribution of immunoreactive Peptide B in the rat brain. Biochem. Biophys. Res. Commun. 139, 1024-1032.

10,404 total views, 1 views today


Warning: Unknown: open(/home/content/83/4542083/tmp/sess_r65tg679hihjju94ajfdiuj880, O_RDWR) failed: No such file or directory (2) in Unknown on line 0

Warning: Unknown: Failed to write session data (files). Please verify that the current setting of session.save_path is correct () in Unknown on line 0